€{|£  ^.  ^.  Hill  lltbrarg 


1.& 


^^ 


S00783823   V 


\ 


THIS  BOOK  IS  DUE  ON  THE  DATE 
INDICATED  BELOW  AND  IS  SUB- 
JECT TO  AN  OVERDUE  FINE  AS 
POSTED  AT  THE  CIRCULATION 
DESK. 


APR^O  199S 


ik 


MAY  1  9  2004 


\ 


\ 


THE  BEHAVIOR  OF  STOMATA 


J.  V.  G.  LOFTFIELD 


..  %'•• 


Published  by  the  Carnegie  Institution  op  Washington 
Washington,    1921 


mi,£  ^.  ^.  pm  pbrarg 


^ortl;  Carolina  ^iah  (HoUcge 

L5 


#f 


CARNEGIE  INSTITUTION  OF  WASHINGTON 
Publication  No.  314 


PRESS  OF  A.  B.  GKAHAM  CO. 
WASHINGTON,  D.  C. 


MC,& 


Zi 


CONTENTS. 


PAGE 

Introduction 7 

I.  Thr  Daily  March  of  Stomatal  Movement IS 

The  daily  movement  in  alfalfa 18 

The  daily  movement  in  potato 27 

The  daily  movement  in  sugar-beet 33 

The  daily  movement  in  onion 37 

The  daily  movement  in  cereals 39 

Discussion  of  results 45 

Summary 48 

II.  Effect  of  Physical  Factors  and  Plant  Conditions  upon  Stomatal  Movement  50 

Light 50 

Temperature 56 

Evaporation  and  causal  factors 59 

"^  Water-content 66 

Leaf  turgor 68 

Plant  habit  and  condition 71 

Summary 74 

III.  Effect  op  Stomatal  Movement  upon  Transpiration 77 

Summary 101 

BiBLIOORAPHY 103 


LIST  OF  ILLUSTRATIONS. 


PLATES. 
Plate  1. 

A.  Wheat  stoma  wedged  open  by  dust. 

B.  Stoma  of  Rumex  jxitientia. 

C.  Epiderm  of  potato,  showing  stomata  in  all  stages  of  development. 
Plate  2. 

A.  Upper  and  lower  epiderm  of  alfalfa. 

B.  llpper  and  lower  epiderm  of  potato. 

Plati5  3.  Scries  10,  showing  condition  of  upper  (outer)  and  lower  (inner)  stomata  of 
potato  at  each  hour  of  a  24-hour  day,  together  with  the  curves  for  sunlight, 
temperature,  and  humidity. 

Plate  4. 

A.  Upper  and  lower  epiderm  of  sugar-beet. 

B.  Epiderm  of  onion. 

C.  Epiderm  of  corn. 
Plate  5. 

A.  Cross-section  of  alfalfa  leaf,  showing  stoma  and  chamber. 

B.  Cross-section  of  sugar-beet  leaf,  showing  stomata  and  chambers. 
Plate  6. 

A.  Cross-section  of  potato  leaf,  showing  stomata,  chambers,  and  air-passages. 

B.  Cross-section  of  corn  leaf,  showing  stomata  and  chambers. 

Plate  7.  Series  11,  showing  upper  and  lower  stomata  of  sugar-beet,  and  factors  during 

a  24-hour  day. 
Plate    8.  Series  16,  showing  stomata  of  onion,  and  factors  during  a  24-hour  day. 
Plate    9.  Series  16,  showing  lower  stomata  of  corn,  and  factors  during  a  24-hour  day. 
Plate  10.  Control  cabinets  used  for  light  experiments  with  stomata. 
Plate  11.  Type  of  potometer  used  for  stomata  experiments. 
Plate  12.  Series  28,  showing   upper  and  lower   stomata    of   Fouquiera  spleridens,  and 

factors  during  a  24-hour  day. 
Pl.ate  13.  Series  32,  showing  upper  and  lower  stomata  of  alfalfa  from  a  heavily  irrigated 

plot,  and  factors  during  a  24-hour  day. 
Plate  14.  Series  32,  showing  upper  and  lower  stomata  of  cut  stems  of  alfalfa,  and  factors 

during  a  24-hour  day. 
Plate  15.  Series  33,  showing   upper  and  lower   stomata  of  cow-beet   in  dry    pots,  and 

factors  during  a  24-hour  day. 
Plate  16.  Serias  33,  showing   upper  and  lower  stomata   of  oow-beot  in  moist    soil,  and 

factors  during  a  24-hour  day. 

FIGURES. 

1.  Three  plants  of  alfalfa  series  14,  stripped  for  27,  17,  and  11  hours  respectively,  show- 

ing that  the  loss  of  a  few  leaves  does  not  afTect  the  stomatal  movement. 

2.  Series  1,  showing  movement  in  upper  stomata  of  alfalfa  and  lower  stomata  of  barley. 

3.  Series  2,  showing  movement  in  upper  stomata  of  alfalfa. 

4.  Series  3,  showing  movement  in  upper  stomata  of  alfalfa. 

5.  Composite  series  4-5,  showing  movement  in  upper  stomata  of  alfalfa. 

6.  Series  26,  showing  movement  in  upper  stomata  of  alfalfa. 

7.  Series  10,  weather  data  for  June  8-9,  1916. 

8.  Series  10,  showing  movement  in  upper,  lower,  and  stem  stomata  of  alfalfa. 

9.  Movement  in  upper  stomata  of  alfalfa,  showing  change  from  day  opening  and  night 

closure  to  night  opening  and  day  clo.sure,  as  evaporation  becomes  more  intense. 

10.  Series  10,  showing  movement  in  upper  and  lower  stomata  of  potato. 

11.  Series  12,  weather  data  for  June  21-22,  1916. 

12.  Series  12,  showing  movement  in  upper  and  lower  stomata  of  potato  and  upper  sto- 

mata of  alfalfa. 

13.  Series  20,  showing  weather  data  for  August  25-26,  1916. 

14.  Series  20,  showing  movement  in  upper  and  lower  stomata  of  potato,  plot  7. 


LIST  OP  ILLUSTRATIONS.  5 

15.  Movement  in  lower  stomata  of  potato  under  (A)  high  water-content  and  moderate 

evaporation,   (B)  low  water-content  and  moderate  evaporation,  and  (C)   low 
water-content  and  excessive  evaporation. 

16.  Series  11,  weather  data  for  June  20-21,  1916. 

17.  Series  11,  showing  movement  in  upper  and  lower  stomata  of  sugar-beet. 

18.  Series  16,  weather  data  for  July  26-27,  1916. 

19.  Series  16,  showing  effect  on  movement  of  reversing  the  leaves  of  sugar-beet. 

20.  Series  16,  showing  movement  in  stomata  of  onion,  and  upper  stomata  of  alfalfa. 

21.  Series  2,  showing  movement  in  lower  stomata  of  oats  and  of  barley. 

22.  Series  3,  showing  movement  in  lower  stomata  of  oats  and  of  barley,  and  upper  sto- 

mata of  alfalfa. 

23.  Series  10,  showing  movement  in  lower  stomata  of  wheat,  barley,  and  oats. 

24.  Series  11,  showing  movement  in  lower  stomata  of  wheat,  oats,  and  barley. 

25.  Series  11,  showing  movement  in  lower  stomata  of  corn  and  millet. 

26.  Series  26,  showing  movement  in  lower  stomata  of  wheat  growing  in  the  greenhouse. 

27.  Series  6,  showing  movement  in  lower  stomata  of  Lombardy  poplar,  and  starch-index 

of  the  guard-cells. 

28.  Series  7,  showing  movement  in  lower  stomata  of  Rumex  patientia,  and  starch-index 

of  the  guard-cells. 

29.  Series  10,  showing  movement  in  upper  stomata  of   alfalfa,  and  starch-index  of  the 

guard-cells. 

30.  Relation  of  speed  of  total  opening  to  temperature. 

31.  Series  34,  factor  data  for  September  10-11,  1919. 

32.  Series  33,  showing  evaporation,  transpiration,  and  the  factors  concerned  for  September 

8-9,  1919. 

33.  Series  33,  showing  evaporation  from  white-cyUnder  |porous-cup  in  cubic  centimeters 

per  hour,  compared  with  product  of  vapor-pressure  deficit  and  wind  velocity, 
calculated  by  Johnston's  method. 

34.  Series  35,  showing  stomatal    movement  in    heavily  watered    plants  of  alfalfa,   the 

partial  closure  at  8  and  10  a.  m.  following  the  disappearance  of  the  dew. 

35.  "Relative  transpiration"  based  on  evaporation  from  blotting-paper  atmometer  and 

from  white-cylinder  porous-cup,  compared  with  stomatal  movement  in  the  onion. 

36.  Effect  of  water-content  on  movement  in  the  upper  stomata  of  alfalfa. 

37.  Series  17,  weather  data  for  August  8-9,  1916. 

38.  Series  17,  showing  average  movement  of  alfalfa  stomata  in  watered  and  unwaterad 

field  plants,  and  in  cut  stems. 

39.  Series  17,  showing  stomatal  movement  and  transpiration  in  cut  stems  of  alfalfa. 

40.  Series  20,  weather  data  for  August  25-26,  1916. 

41.  Series  20,  showing  movement  in  upper  and  lower  stomata  of  potted  potato  plants, 

and  in  upper  and  lower  stomata  of  cut  potato  stems. 

42.  Series  20,  showing  movement  in  upper  and  lower  stomata  of  heavily  watered  potato 

plants,  and  in  upper  and  lower  stomata  of  plants  in  very  dry  soil. 

43.  Series  20,  showing  average  stomatal  movement  and  transpiration  in  cut  stems  of  potato. 

44.  Series  29,  weather  data  for  March  15-16,  1918. 

45.  Series  29,  showing  average  movement  in  upper  and  lower  stomata  of  watered  and 

unwatered  field  plants  and  in  cut  stems  of  Fouquiera  splendens. 

46.  Series  29,  showing  average  movement  in  upper  and  lower  stomata  of  field  plants, 

and  cut  stems  of  Verbena  ciliata. 

47.  Series  29,  showing  stomatal    movement  and  transpiration  in  cut  stems  of  Fmiquiera 

splendens. 

48.  Series  29,  showing  stomatal  movement  and  transpiration  in  cut  stems  of  Verbena  ciliata. 

49.  Series  32,  weather  data  for  August  25-26,  1919. 

50.  Series  32,  showing    stomatal   movement  averaged  for   2-hour  periods,   and  trans- 

piration in  miUigrams  per  minute  for  the  same  periods  in  heavily  watered  potted 
plants  of  alfalfa. 

51.  Series  32,  showing  stomatal    movement   averaged   per   2-hour   period,    and   trans- 

piration in  milUgrams  per  minute  of  dry  alfalfa  phytometers. 

52.  Series  32,  showing  transpiration  of  wet  and  dry  alfalfa  phytometers. 

53.  Series  33,  showing  average  movement  of  upper  and  lower  stomata,  and  transpiration 

of  cow-beets  in  dry  pots. 

54.  Series  33,  showing  transpiration  from  potted  cow-beets  in  dry  pots  and  from  potometers. 


THE  BEHAVIOE  OP  STOMATA. 


INTRODUCTION. 

This  investigation  was  undertaken  to  discover  what  changes 
take  place  in  the  apertures  of  stomata  throughout  the  day  and 
night,  the  influence  of  physical  factors  upon  such  changes,  and  the 
final  effect  upon  transpiration.  Hence  the  investigation  falls 
naturally  into  three  subdivisions  which  form  the  sections  of  this 
volume.  As  previous  investigators  have  usually  worked  with  few 
plants  and  in  one  region,  it  was  felt  that  considerable  light  could 
be  thrown  upon  the  subject  by  using  many  plants  and  working  in 
several  regions  of  diverse  climate.  Experiments  which  were  first 
carried  out  at  the  American  Smelting  and  Refining  Company's 
laboratory  near  Salt  Lake  City  were  therefore  continued  at  the 
Desert  Laboratory  at  Tucson,  Arizona,  and  at  the  University  of 
Minnesota,  Minneapolis,  Minnesota,  as  well  as  at  Salt  Lake  City. 
A  wide  range  of  climate  was  thus  available,  since  the  spring  climate 
of  Minneapolis  is  rather  humid,  the  summer  climate  of  Salt  Lake 
City  dry  and  sometimes  hot,  and  the  winter  in  Tucson  dry  and  warm. 
For  a  number  of  reasons,  most  of  the  experiments  were  made  at  the 
Salt  Lake  laboratory,  where  the  water-content  could  be  controlled 
readily  and  the  range  of  humidity  and  temperature  from  day  to  day 
was  almost  as  great  as  in  the  desert. 

At  the  outset,  careful  consideration  was  given  the  methods  hitherto 
employed  to  determine  changes  in  stomatal  apertures.  The  horn 
and  yucca  hygroscopes  used  by  Darwin  (1898)  were  properly 
criticized  by  Lloyd  (1908)  as  measures  of  transpiration  rather  than 
of  the  condition  of  the  stomata.  Cobalt-chloride  paper  is  open  to 
the  same  objection,  and  therefore  only  indirectly  throws  light 
upon  the  opening  of  the  stomata.  Petroleum  ether,  when  dropped 
upon  a  leaf,  passes  through  it  if  the  stomata  are  open  and  some- 
times gives  it  a  water-soaked,  translucent  appearance.  Hence  this 
reagent  may  be  used  as  a  qualitative  test  for  stomatal  opening. 
These  methods,  however,  are  no  longer  in  use,  and  this  left  but 
three  methods  to  be  considered  for  the  purpose  of  the  investigation. 

The  two  methods  used  by  Lloyd  (1908,  1913)  may  be  regarded  as 
reliable  in  a  high  degree.  The  first  method  consists  in  observing 
the  degree  of  opening  in  stomata  in  properly  fixed  strips  of  epiderm, 
the  second  in  observing  the  stomata  in  their  natural  state  on  the 
leaves  while  these  are  still  on  the  plant.  Naturally,  the  latter 
method  is  preferable,  since  it  shows  the  stomata  on  leaves  which 
are  undisturbed  until  they  are  placed  under  the  microscope.     But 


8  BEHAVIOR   OF   STOMATA. 

the  impossibility  of  doing  this  at  night  or  in  poor  light  reduces  the 
applicability  of  this  method  very  considerably.  The  porometer 
method,  devised  by  Darwin  and  Pertz  (1911)  and  modified  and 
improved  by  Laidlaw  and  Knight  (1916),  was  not  considered 
favorably  for  several  reasons,  chief  among  which  were  the  unnatural 
conditions  introduced  by  pulling  a  current  of  air  through  the  inter- 
cellular spaces  of  the  leaf,  and  a  doubt  as  to  what  porometer  readings 
actually  represented.  To  the  best  of  the  writer's  knowledge,  no 
comparison  has  been  made  between  a  series  of  direct  observations 
upon  the  condition  of  the  stomata  and  porometer  readings  made 
at  the  same  time.  Until  the  method  is  checked  in  this  manner, 
it  is  felt  that  its  reliability  is  questionable. 

For  the  reasons  given,  it  was  felt  that  the  most  desirable  method 
to  employ  was  the  first  one  used  by  Lloyd  (1908).  This  consists 
in  stripping  the  epiderm  from  the  leaf  and  quickly  plunging  it  into 
absolute  alcohol,  which  immediately  fixes  the  epidermal  cells, 
keeping  them  in  the  shape  they  were  in  when  immersed.  The 
guard-cells  can  not  lose  an  appreciable  amount  of  water  in  the 
fraction  of  a  second  between  stripping  and  immersion  in  alcohol. 
The  effect  of  the  alcohol  is  to  dehydrate  the  cell- wall  before  pen- 
etrating the  cell  proper  to  any  extent.  The  dehydration  of  the 
cellulose  wall  causes  it  to  become  very  stiff  and  hard.  As  a  con- 
sequence the  cells  retain  their  original  form  in  spite  of  the  fact  that 
the  alcohol  next  removes  the  water  from  within  the  cell.  This 
form  is  maintained  for  some  time,  provided  the  walls  are  kept 
dehydrated.  Theoretically,  therefore,  the  method  seemed  sound,  but 
it  was  evident  that  it  could  not  be  considered  reliable  until  checked 
by  direct  observation  of  the  stomata  of  living  leaves,  not  only  for 
each  species  and  variety  of  plant  studied,  but  for  a  wide  range  of 
stomatal  apertures  for  each  as  Avell.  Not  only  was  this  done,  but 
in  addition  several  parallel  series  were  made  with  the  more  important 
plants,  one  series  being  the  usual  set  of  epidermal  strips  collected 
each  hour,  the  other  a  set  of  measurements  of  stomatal  apertures 
on  the  living  leaves  of  the  same  plant,  also  made  each  hour. 

The  check  upon  the  method  was  ordinarily  made  in  the  following 
fashion:  A  stand  for  the  microscope  was  erected  in  such  a  manner 
that  a  leaf  could  be  brought  into  the  field  with  the  least  possible 
change  of  position  and  consequent  disturbance.  This  leaf  was  then 
lightly  clamped  between  a  long  cover-glass  and  slide,  and  about  10 
stomata  quickly  measured.  Not  more  than  2  minutes  was  allowed 
for  this  operation,  in  order  to  prevent  any  serious  change  while  the 
leaf  was  exposed  to  these  unnatural  conditions.  Then  the  leaf  was 
released  and  stripped  in  the  usual  manner,  great  care  being  exercised 
to  secure  the  area  just  examined.     After  the  customary  two  days  of 


INTRODUCTION. 


9 


staining,  a  similar  number  of  stomata  were  measured  in  the  strip  and 
the  results  compared.  Table  1  gives  in  millimeters  the  results  obtained 
for  alfalfa,  which  are  typical  of  those  obtained  with  other  plants. 


Table  1.— 

'iesidts  obtained  from  alfalfa 

• 

Leaf  11,  upper 

Leaf  22,  upper 

Leaf  22,  lower 

Leaf  25,  upper 

Leaf  25,  lower 

surface. 

surface. 

surface. 

surface. 

surface. 

Leaf. 

Strip. 

Leaf. 

Strip. 

Leaf. 

Strip. 

Leaf. 

Strip. 

Leaf. 

Strip. 

8.5  X    2.2 

8.0  X  2.5 

8.5  X  2.5 

8.0  X  2.5 

7.0  X  2.5 

6.0  X1.3 

8.5  XO.l 

8.0  X  0.0 

7.0  X2.1 

7.0  X  0.0 

8.0  X    3.0 

7.0  X  3.0 

8.0  X  3.0 

7.0  X  3.0 

7.0  X  2.4 

7.0  X  2.9 

8.0  X  0.0 

9.0  X0.5 

7.5  XO.O 

7.0  X  0.5 

6.3  X    3.0 

6.5  X  2.4 

6.5  X  3.0 

9.0  X4.0 

7.0  X2.1 

7.0  X  2.6 

7.4  X  0.0 

8.5  XO.O 

7.2  X  0.0 

6.0  X0.2 

8.1  X    2.5 

8.2  X  2.0 

8.0  X2.4 

8.0  X  2.0 

8.0  X  2.9 

6.0  Xl.l 

7.0  X0.4 

7.5  X  0.0 

7.2  X  1.0 

6.4  X  1.0 

8.0  X    2.5 

7.0  X  2.9 

8.0  X  2.9 

7.0  X  3.0 

7.0  X  2.6 

6.5  X  1.8 

8.0  X0.2 

8.0  X0.2 

7.5  X0.2 

7.0  X0.2 

60  X    2.3 

7.7  X3.2 

6.0  X  2.0 

8.0  X2.9 

5.0  X  2.0 

8.5  X  3.0 

8.0  X0.2 

6.5  X0.5 

8.0  X0.5 

7.0  X  1.0 

7.0  X    2.5 

8.0  X  3.5 

7.1  X2.4 

8.0  X  2.9 

8.0  X2.3 

8.2  X2.8 

8.0  X  0.4 

6.5  X  1.0 

7.0  X0.4 

6.0  X0.2 

7.0  X    3.0 

6.0  X  2.0 

9.0  X3.0 

6.0  X  2.2 

6.0  X2.1 

5.0  X  2.0 

6.5  X0.4 

8.0  X  0.4 

7.0  X  0.3 

6.5  X0.4 

8.0  X    3.0 

9.0  X  2.8 

7.9  X  2.9 

8.0  X  2.0 

7.0  X  2.0 

8.0  X  3.0 

9.0  X  0.4 

7.5  X0.5 

7.0  X  0.6 

8.0  X0.2 

8.5  X    3.0 

8.0  X  3.0 

8.5  X3.1 

8.2  X3.0 

8.0  X  2.0 

7.0  X  2.6 

6.0  Xl.O 

8.0X0.2 

8.0  X0.4 
7.6  XO.O 
6.0  X0.2 

7.0  X  2.0 
8.0  X  0.0 
8.0  X  0.2 

76.4      27.0 

75.4    27.3 

77.5     27.2 

77.2    27.5 

70.0     22.7 

69.2     23.1 

76.4       3.1 

77.5      3.3 

93.0       6.0 

91.9      6.1 

Aver 

age 

Aver 

age 

Aver 

age 

Aver 

age 

Aver 

age 

7.5  X    2.7 

7.5  X  2.7 

7.7  X  2.7 

7.7  X  2.7 

7.0  X  2.3 

6.9  X  2.3 

7.6  X  0.3 

7.7X0.3 

7.2X0.5 

7.1  X0.5 

Lower  surface. 

Leaf.          Strip. 

Closed.     Closed. 

The  results  from  alfalfa  are  given,  since  most  of  the  experiments 
discussed  were  performed  upon  alfalfa.  At  first  this  was  due  to 
the  fact  that  it  was  the  most  important  plant  susceptible  to  SO2 
injury.  Later,  its  sensitive  stomata  and  great  availability  made  it 
very  useful  in  many  of  the  series  as  a  basis  of  comparison  in  the 
study  of  the  daily  march  of  stomatal  movement  in  other  plants. 
The  results  from  the  checks  did  not  always  agree  as  well  as  in  the 
above  tables,  but  the  agreement  became  greater  as  the  number  of 
stomata  measured  was  increased.  The  reason  for  trying  to  get  as 
nearly  the  same  area  as  possible  in  stripping  was  not  because  of 
any  hope  of  getting  the  same  stomata  measured  in  the  strip  as  on 
the  leaf,  but  because  the  effect  of  the  great  variation  that  often 
exists  in  the  stomata  on  the  same  leaf  can  best  be  avoided  in  this 
manner.  Table  2,  showing  the  least  amount  of  agreement  in  the 
two  sets  of  measurements  of  those  made  on  alfalfa,  will  give  the 
reader  a  clearer  idea  of  what  is  meant. 

In  table  2  the  agreement  is  not  at  all  close.  The  second  set  of 
10  measurements  of  stomata  in  the  strip  is  closer  than  the  first  10, 
but  when  the  two  sets  are  averaged  together,  the  result  is  7.1  by 
0.39,  which  is  still  nearer  the  average  for  width  of  pore.  Had  20 
stomata  been  measured  in  the  leaf,  the  agreement  would  probably 
have  been  quite  close.     Where  there  was  much  variation  in  size 


10 


BEHAVIOR   OF   STOMATA. 


of  stomatal  apertures  the  number  of  measurements  was  usually 
increased,  when  the  agreement  was  invariably  much  better. 

Many  objections  have  been  raised  to  the  stripping  method  since 
its  development  by  Lloyd,  most  of  them  made  without  sufficient 
reason.  The  essence  of  the  method  is  the  almost  instant  dehydra- 
tion by  the  alcohol  and  consequent  hardening  of  the  cellulose  walls 

Table  2. 


Leaf  13,  lower  epiderm. 

Leaf. 

Strip  L 

Strip  II. 

7.3  X  0.2 
6.0  X  0.2 
6.2  X  0.2 
6.0  X  1.0 
7.0  X  0.4 

6.4  X  1.0 
7.0  X  0.2 
7.2  X  0.2 
7.4  X  0.2 
7.6  X  0.0 

8.0  X  0.5 
7.0  X  0.4 
7.5  X  0.0 
7.0  X  0.2 
7.0  X  0.0 
7.0  X  0.0 
7.5  X  0.2 
7.5  X  0.2 
8.0  X  0.2 
8.0  X  0.4 

6.5  X  0.4 
8.0  X  0.2 
7.0  X  2.0 
6.0  X  0.2 
7.0  X  1.0 
7.0  X  0.2 
6.4  X  1.0 
6.0  X  0.2 
7.0  X  0.5 
7.0  X  0.0 

68 . 1        3.6 
Av.  6.8  X  0.46 

74.5        2.1 
7.5  X  0.21 

67.9        5.7 
6.8  X  0.57 

of  the  guard-cells  and  surrounding  epidermal  cells,  which  fixes  them 
in  the  shape  in  which  they  were  when  plunged  into  the  alcohol.  The 
dilution  of  alcohol  in  the  cell-wall  caused  by  the  extraction  of  the 
water  within  the  cell  is  not  sufficient  to  soften  these  walls  or  to  cause 
them  to  lose  their  form.  The  error,  if  error  there  be,  must  occur 
during  stripping  or  immediately  following.  The  objection  made  by 
I^aidlaw  and  Knight  that  the  jarring  due  to  the  operation  causes 
closure  may  be  dismissed,  for,  if  stomata  of  plants  growing  in  the 
open  field  closed  each  time  they  were  jarred,  they  would  be  perma- 
nently closed  in  a  region  of  almost  constant  wind,  such  as  the  Great 
Plains  or  the  Great  Basin.  Moreover,  stripping  itself  would  make  it 
impossible  to  find  any  strips  with  open  stomata. 

The  more  common  objection  that  shrinkage  and  distortion  occur 
is  to  some  extent  true.  A  large  number  of  strips  were  measured  to 
find  the  amount  of  this  shrinkage,  and  it  was  found  to  be  less  than  1 
per  cent  when  it  occurred  at  all.  Immediately  after  stripping,  the 
width  and  length  of  the  stripped  area  upon  the  leaf  were  measured 
with  dividers  and  compared  with  the  length  and  width  of  the  strip. 
In  most  cases  the  shrinkage  was  not  perceptible,  but  in  the  case 
of  wilted  leaves  there  was  very  slight  shrinkage.  But  as  this  was 
always  less  than  1  per  cent,  the  greatest  error  possible  was  not  more 


INTRODUCTION.  11 

than  0.04  micron  in  the  width  of  an  alfalfa  stoma,  while  the  smallest 
division  that  can  be  accurately  measured  is  0.2  micron. 

The  distortion  which  occurred  as  a  result  of  the  epidermal  strip 
rolUng  or  curling  was  usually  caused  by  keeping  the  strip  exposed  to 
the  air  too  long.  As  this  shrinking  was  greater  on  the  one  side  than 
on  the  other,  and  greater  in  one  direction  than  the  other,  it  was 
thought  possible  that  the  stomata  lying  in  the  direction  of  the  axis 
of  rolling  would  tend  to  close,  and  those  lying  at  right  angles  to  this 
direction  would  tend  to  open  slightly.  If  this  occurred  it  was  too 
slight  to  be  detected.  Nevertheless,  such  strips  were  not  used, 
except  in  rare  cases,  not  so  much  because  of  doubt  in  regard  to  their 
accuracy  as  because  of  difficulty  in  photographing  them.  The 
curled  strips  apparently  checked  just  as  closely  as  the  others  with 
measurements  of  the  stomata  in  situ. 

The  chief  reason  for  confidence  in  the  method  lies  in  the  agreement 
of  the  stomatal  openings  observed  in  the  undisturbed  leaves  with 
those  measured  in  the  strips  removed  at  the  same  time.  Any  serious 
objection  to  the  method  must  come  through  showing  that  there  is 
no  such  agreement  between  results  from  properly  performed  stripping 
and  those  obtained  by  carefully  conducted  observations  of  stomata 
in  situ.  Except  for  the  impossibility  of  observing  them  during  the 
hours  of  darkness  and  poor  light  without  the  use  of  strong  artificial 
light,  this  latter  method  might  have  been  used  throughout.  How- 
ever, other  objections  were  decisive,  in  view  of  the  objects  sought. 
These  were  the  impossibility  of  photographing  the  stomata  in  such 
leaves,  and  the  difficulty  of  finding  enough  leaves  on  a  single  plant 
in  such  position  that  they  could  be  slipped  under  the  microscope 
without  undue  disturbance. 

It  must  be  emphasized  that  an  examination  of  a  leaf  in  this  man- 
ner does  set  up  a  disturbance  which  sooner  or  later  appears  in  the 
stomata,  and  hence  such  a  leaf  can  be  used  but  once  in  the  series. 
This  was  tested  several  times  by  comparing  such  a  leaf  with  some 
undisturbed  leaf  half  an  hour  or  an  hour  later.  The  usual  effect  was 
to  produce  closure  of  the  stomata.  Leaving  a  leaf  under  the  micro- 
scope would  in  extreme  cases  cause  closure  in  3  minutes  in  some  sto- 
mata, but  as  a  rule  10  minutes  or  even  more  elapsed  before  a  change 
occurred.  As  the  experiments  demonstrated  that  the  stripping 
method  was  as  reliable  as  direct  observations,  it  was  used  because  of 
its  greater  convenience,  and  direct  examination  merely  for  checking 
and  comparison. 

The  accuracy  of  the  method  depends  greatly  upon  the  process  of 
stripping.  To  gain  speed,  the  scalpel  used  in  making  the  cut  to 
start  the  strip,  and  the  forceps  used  in  seizing  the  epiderm  and  in 
stripping,  were  bound  together  with  rubber  bands,  the  end  of  the 


12  BEHAVIOR   OF   STOMATA. 

forceps  projecting  about  an  inch  beyond  the  tip  of  the  scalpel  blade. 
The  cut  once  made,  the  instrument  was  twirled  half  a  turn  to  bring 
the  forceps  into  position,  the  cut  end  seized,  and  the  epiderm  plunged 
into  the  previously  opened  vial  of  alcohol  well  within  a  second.  A 
good  flash-light  was  usually  used  for  night  work,  or  at  times  an  elec- 
tric light  from  a  field  socket  nearby.  This  light  was  sufficient  to 
select  the  leaves  desired  and  to  strip  them,  the  process  taking  about 
a  minute,  an  interval  too  short  to  cause  opening  by  the  light  used. 

A  number  of  precautions  must  be  observed  in  stripping  if  the 
results  are  to  be  reliable.  Stomata  vary  greatly  in  sensitiveness  in  re- 
sponse to  stripping.  Those  of  cereals  and  grasses  generally  are  very 
sensitive  to  exposure  to  air  and  must  be  plunged  into  the  alcohol  in 
less  than  a  second,  while  those  of  potato  or  Rumex  patientia  may  be 
kept  out  for  a  much  longer  time.  A  strip  of  this  latter  was  kept  ex- 
posed to  the  air  for  a  full  minute  in  one  experiment  without  change 
of  stomatal  opening.  Corn  is  not  so  sensitive  as  wheat  or  barley  to 
such  exposure,  but  even  with  this  plant  it  is  best  to  reduce  the  process 
to  less  than  2  seconds  when  stripping.  Another  precaution  is  not  to 
strip  a  wet  leaf.  If  rain  or  heavy  dew  has  fallen,  the  moisture  should 
be  removed  with  filter-paper  or  absorbent  cotton,  since  the  water 
carried  with  the  strip  may  dilute  the  alcohol  seriously.  The  water 
on  the  strip  may  also  affect  the  stomata  before  the  strip  reaches  the 
alcohol,  especially  when,  as  is  usually  the  case,  capillarity  causes  the 
water  to  be  pulled  to  the  under  side  of  the  strip.  This  latter  objec- 
tion depends  a  good  deal  upon  the  kind  of  plant  stripped.  The  epi- 
derm of  alfalfa,  which  has  sensitive  stomata,  has  been  kept  in  water 
for  an  hour  or  more  after  stripping  and  no  appreciable  difference  found 
as  compared  with  a  similar  strip  plunged  into  alcohol  in  the  usual 
manner.  However,  even  with  alfalfa,  the  water  carried  with  the 
strip  will  often  dilute  the  alcohol  in  contact  with  the  strip  long 
enough  to  cause  partial  or  complete  collapse  of  the  guard-cells. 

Great  variation  in  the  size  of  stomatal  apertures  on  various  leaves 
may  be  found  at  any  one  time,  not  only  on  leaves  of  different  indi- 
viduals, but  also  those  of  the  same  plant.  They  even  vary  much 
at  times  on  the  same  leaf.  The  causes  for  this  are  many.  No  two 
plants  are  found  in  exactly  similar  situations,  nor  are  the  leaves  on 
the  same  plant  under  identical  conditions.  The  usual  cause  for 
difference  in  stomatal  openings  in  the  various  leaves  on  the  same 
plant  lies  in  their  age  or,  more  properly,  their  degree  of  maturity. 
A  leaf  does  not  have  functional  stomata  until  it  is  fully  grown,  and 
young  stomata  do  not  open  so  soon  or  stay  open  so  long  as  the 
stomata  on  the  more  mature  leaves.  The  same  is  true  in  even  greater 
measure  of  an  aging  leaf.  Long  before  an  old  leaf  turns  yellow  and 
falls,  the  stomata  have  ceased  to  function  and  remain  permanently 


INTRODUCTION.  13 

closed.  With  experience  one  learns  which  leaves  are  mature  and 
actively  functional,  and  these  alone  should  be  used  in  a  series. 
Another  cause  of  variation  in  the  leaves  on  a  plant  is  due  to  the  fact 
that  the  stomata  of  leaves  that  are  shaded  are  often  closed  when 
those  of  unshaded  leaves  are  open,  or  open  when  the  ones  on  the 
upper  leaves  are  closed.  But  when  enough  stomata  are  measured 
to  insure  checking  out  the  extremes,  the  degree  of  opening  in  the 
mature  leaves  of  the  same  plant  exposed  about  equally  to  the  light 
agree  very  well.  Usually  three  leaves  of  each  set  of  plants  were 
stripped  each  hour,  giving  three  strips  of  epiderm  for  each  leaf- 
surface  for  each  hour.  This  insured  against  loss  due  to  inadvertently 
stripping  an  immature  or  aged  leaf.  Such  an  occurrence  was  very 
rare  after  some  experience  had  been  gained  in  selecting  the  proper 
leaves.  It  also  served  as  a  check  upon  similarity  of  stomatal  behavior 
in  mature  leaves.  The. causes  of  variations  of  the  stomatal  opening 
of  different  plants  will  be  dealt  with  later. 

Twenty-four  hour  series  were  made  of  more  than  60  species  of 
plants,  including  crop,  garden,  and  forage  plants,  weeds,  native 
herbs,  trees,  and  shrubs.  The  more  important  of  these  were  checked 
in  the  same  manner  as  alfalfa,  in  order  to  determine  the  reliability 
of  the  stripping  method.  A  great  many  were  used  in  but  one  series, 
which  was  made,  however,  at  the  same  time  as  an  alfalfa  series,  in 
order  to  determine  the  daily  march  of  its  stomatal  movement  in 
relation  to  a  plant  whose  reaction  to  the  environmental  factors  was 
more  or  less  known.  Except  in  the  case  of  leaves  with  but  one 
stomatal  surface,  each  series  consisted  of  two  sets  of  epiderm,  one 
for  each  surface. 

Few  investigators  seem  to  have  made  long,  continuous  series. 
The  discovery  that  the  variation  in  the  march  of  stomatal  movement 
from  day  to  day  was  considerable  made  it  evident  that  only  a  long, 
continuous  series  would  be  of  any  real  value.  A  group  of  short  series 
covering  the  entire  period  of  the  day  and  night,  but  not  made  within 
the  same  24-hour  period,  fails  to  reveal  the  actual  sequence,  but  makes 
an  artificial  one  having  slight  relationship  to  the  stomatal  movement 
upon  any  such  day.  Because  of  this,  the  usual  series  was  of  24  hours' 
duration.  A  number  of  shorter  ones  were  made  for  special  purposes, 
especially  for  what  may  be  termed  a  reconnaissance  of  the  field  and 
for  investigations  on  the  effect  of  certain  factors.  Longer  series  were 
also  made  quite  frequently,  the  longest  being  continuous  for  68  hours, 
but  the  strain  placed  upon  the  investigator  by  so  long  a  series  is  not 
justified  by  the  results  obtained.  Even  a  24-hour  series  called  for  at 
least  36  hours  of  continuous  labor,  as  it  required  many  hours  of  work 
to  get  everything  in  readiness,  and  several  hours  after  the  series  was 
finished  to  complete  the  work. 


14  BEHAVIOR   OF   STOMATA. 

The  question  of  the  time  of  starting  and  ending  a  series  was  also 
of  importance.  It  was  especially  desirable  that  no  gap  should  occur 
between  the  first  and  last  strip  collected.  If  a  series  began  with  the 
stomata  closed  and  ended  with  them  closed,  the  course  of  events 
leading  to  the  opening  and  final  closing  could  be  followed  throughout, 
but  if  it  began  with  them  closed  and  ended  with  them  open,  the  time 
of  closure  would  be  in  doubt,  and  the  series  left  without  a  definite 
ending.  At  first  it  was  thought  that  the  best  time  to  start  a  series 
would  be  just  before  dawn,  as  the  stomata  would  have  all  night  to 
reach  closure.  Experience  showed,  however,  that  just  after  sunset 
was  the  best  time,  for  if  the  stomata  closed  at  all  they  usually  closed 
at  this  time. 

Several  days  prior  to  photographing,  the  strips  were  transferred 
to  vials  containing  a  saturated  solution  of  Congo  Red  in  absolute 
alcohol.  This  stains  the  cell- walls  chiefly,  making  them  stand  out 
very  clearly.  A  number  of  other  stains  were  tried,  but  none  proved 
as  satisfactory  as  Congo  Red.  When  ready  for  photographing,  the 
strip  was  trimmed  and  placed  upon  a  special  slide  with  two  spring 
clips  which  held  the  cover-glass  in  place,  and  then  mounted  in  absolute 
alcohol.  This  type  of  slide  was  not  absolutely  necessary,  but  it  was 
very  convenient,  because  it  prevented  the  strip  from  floating  and  the 
consequent  loss  of  a  clear,  sharp  image.  Because  of  the  alcohol 
mount,  an  upright  camera  was  used.  This  consisted  of  a  stand  for  the 
microscope,  a  hood  which  was  tied  around  the  top  of  the  microscope, 
a  camera-box  taking  a  5  by  7  plate-holder,  and  a  frame  which  sup- 
ported all  of  these.  A  Spencer  microscope  was  used  with  a  4  mm. 
objective,  a  Zeiss  projection  ocular  No.  2  being  used  to  project  the 
micrograph  upon  the  photographic  plate.  The  same  microscope  and 
camera  were  used  for  all  microphotographs  made.  The  magni- 
fication (345  diameters)  was  the  largest  that  could  be  employed  and 
still  have  the  entire  field  upon  a  5  by  7  plate. 

The  photographs  of  a  series  were  mounted  for  ready  comparison 
on  charts  54  inches  square,  in  the  form  of  a  24-hour  clock.  Those 
reproduced  are  reduced  charts  made  by  cutting  out  an  average  stoma 
or  group  of  stomata  from  each  photograph  and  mounting  them  on  a 
chart  8  inches  in  diameter.  The  light,  temperature,  and  relative 
humidity  curves  are  given  in  a  circular  graph  inside  the  circle  of 
microphotographs.  Whenever  this  circle  is  a  double  one,  the  outer 
circle  shows  the  upper  epiderm  and  the  inner  circle  the  lower  epiderm 
of  the  leaves  of  the  plant  under  consideration.  In  the  greater  number 
of  series  the  strips  were  not  photographed,  but  about  20  stomata  of 
each  were  measured  and  averaged  to  get  the  material  for  stomatal 
graphs.  All  the  charts  are  not  reproduced  here,  since  that  would 
involve  needless  duplication  and  bring  in  species  that  can  not  be 
discussed  at  this  time. 


INTRODUCTION.  15 

The  physical  data  were  obtained  chiefly  by  means  of  simple  instru- 
ments. A  stop-watch  photometer  was  used  for  the  light  data,  a 
cog  psychrometer  to  determine  the  relative  humidity,  a  compen- 
sated aneroid  barometer  for  obtaining  the  pressure,  a  group  of  ordi- 
nary chemical  thermometers  checking  within  a  tenth  of  a  degree 
centigrade  to  determine  the  temperature  at  various  levels  of  the  soil 
and  air,  and  a  Tycos  anemometer  to  measure  the  velocity  of  the 
wind.  The  water-content  was  determined  by  taking  soil  samples  at 
the  depths  described,  weighing,  drying  them  on  a  water-bath,  and 
weighing  again  after  they  had  become  constant  in  weight.  Samples 
were  always  taken  in  duplicate. 

The  stop-watch  photometer  devised  by  Clements  may  need  a  fuller 
description.  It  consists  of  a  drum  on  which  a  strip  of  SoUo  paper  is 
wound  and  inclosed  in  a  case  which  is  light-proof.  This  case  has  a 
rectangular  opening  directly  over  the  strip  of  Solio.  This  opening  is 
covered  and  light-proof  until  an  exposure  is  desired.  The  case  can 
be  revolved  one  twenty-fifth  of  the  circumference  over  the  drum  and 
paper,  exposing  a  new  area  on  the  strip  each  time.  The  slide  is  so 
arranged  that  when  the  opening  in  the  case  is  uncovered  the  stop- 
watch attached  to  the  front  of  the  photometer  is  started,  and  stopped 
when  the  slide  is  closed.  In  this  manner  the  time  of  exposure  can 
be  measured  accurately  to  a  fifth  of  a  second — in  fact,  much  more 
accurately  than  a  fifth,  for  the  operator  soon  learns  to  snap  the  slide 
shut  just  at  the  moment  that  the  hand  of  the  watch  jumps  to  the 
proper  fifth  of  a  second.  The  time  is  probably  accurate  to  a  fiftieth 
of  a  second.  The  slide  is  attached  to  a  spring  which  instantly  covers 
the  opening  the  moment  it  is  released. 

The  standard  was  made  at  noon  on  a  clear  day  at  the  time  of  the 
summer  solstice.  This  consisted  of  a  Solio  strip  exposed  1  second 
at  the  first  area,  2  seconds  at  the  second  area,  3  seconds  at  the  third, 
etc.,  the  last  area  being  exposed  25  seconds.  As  a  rule,  several  of 
these  were  made,  one  for  each  clear  day  during  the  proper  period. 
The  one  showing  the  deepest  color  was  selected  as  the  standard. 
When  a  light  reading  was  desired,  an  exposure  was  made  and  the 
time  and  number  of  the  exposure  recorded.  When  the  25  exposures 
were  exhausted,  the  photometer  was  taken  to  the  dark-room,  the 
strip  removed,  and  each  area  of  exposure  compared  with  the  standard. 
When  an  area  exposed  20  seconds  has  the  same  depth  of  color  as  the 
3-second  area  on  the  standard,  the  light  at  that  time  was  three- 
twentieths  of  the  maximum  for  that  region,  or  15  per  cent. 

With  the  exception  of  Lloyd,  no  investigator  seems  to  have  used 
plants  growing  in  a  natural  environment.  In  spite  of  this,  many  of 
these  workers  have  tried  to  apply  their  data  to  plants  growing  natu- 
rally, without  due  allowance  for  the  conditions  under  which  their 
experiments  were  performed.     Gray  and  Peirce  (1917)  experimented 


16 


BEHAVIOR   OP   STOMATA. 


20 


with  plants  grown  in  a  greenhouse  under  shaded  glass  and  in  wooden 
boxes  or  flats,  which  they  state  was  necessary  in  their  cHmate,  and 
assumed  that  such  plants  compare  in  reaction  with  similar  plants 
grown  in  the  field.  The  use  of  a  photometer  would  have  shown 
this  assumption  to  be  unwarranted,  as  fully  50  per  cent  of  the  light  is 
removed  in  a  greenhouse  with  clean  panes.  In  the  case  of  panes 
only  hghtly  frosted,  it  is  probable  that  less  than  15  per  cent  of  the 
light  incident  actually  reached  the  plants.     As  their  experiments 

A 


/ 

/  4^h 

7        I      Zr 

-.t        X   7  X 

Z          X  t  A^ 

7             47        S 

t            XL          ^ 

1.               ^1            \ 

%          ^          /                                ^ 

^^       y'\      /                                      \_ 

8 

9 

10 

'< 

MT 

•     ' 

2 

3 

■4- 

5 

6 

7 

8 

9 

10 

II 

/ 

/ 

1 

/ 

V 

/ 

/ 

\ 

/ 

/ 

\ 

/ 

\ 

/ 

\ 

/ 

\ 

/ 

\ 

1 

\ 

NOON 
100 

90 

80 

70 

60 

50 

4-0 

30 

20 

10 


1234-56789     10 


/ 

\ 

/ 

\ 

i 

/ 

\ 

\  / 

\ 

/ 

V 

7 

\ 

\ 

\ 

6     7     8      9      10     U  NOON  I      234-56789      10       NOON  I       23     4-56789     «0 

B  C 

Fig.  1. — Three  plants  of  alfalfa  series  14,  stripped  for  27,  17,  and  11  hours  respectively,  showing 
that  the  loss  of  a  few  leaves  does  not  affect  the  stomatal  movement. 

were  carried  out  with  a  view  of  discovering  the  effect  of  light  upon 
the  stomatal  movement  of  their  plants,  they  can  apply  only  to  plants 
grown  under  similar  conditions  as  to  light  intensity.  With  plants 
growing  in  a  desert,  water  is  the  factor  which  determines  their  exist- 
ence. In  shade,  even  if  only  partial,  the  determining  factor  is 
light;  hence,  in  experiments  conducted  as  were  theirs,  it  is  to  be 
expected  that  the  stomata  would  respond  to  light  much  more  quickly 
than  to  any  other  factor.     Moreover,  their  terms  "light,"  "hazy," 


INTRODUCTION.  17 

"rather  dark,"  "too  dark  to  see  clearly,"  are  merely  descriptive  and 
can  not  serve  as  measures  of  light. 

In  these  studies,  field  plants  grown  under  natural  conditions  were 
used  exclusively.  All  possible  factors  were  accurately  measured 
and  the  correlations  were  drawn  with  due  regard  to  the  limits  placed 
by  lack  of  data  on  other  reactions  by  the  plant.  Too  much  empha- 
sis can  not  be  placed  on  the  importance  of  having  the  plant  under  as 
normal  conditions  as  possible.  Of  course,  a  plant  on  which  one  leaf 
after  another  is  removed  is  not  entirely  under  normal  conditions,  but 
an  alfalfa  plant  with  approximately  1,000  leaflets  is  not  very  much 
affected  by  the  removal  of  50  during  a  24-hour  series.  This  is  shown 
by  a  series  started  at  8  p.  m.  on  June  26,  1916.  A  large  plant  of  al- 
falfa was  stripped  during  the  night  as  well  as  the  next  day  at  6  a.  m., 
June  27;  another  set  was  added  to  the  series  by  stripping  a  plant  close 
by,  and  hence  under  as  nearly  the  same  conditions  as  possible  to  the 
first  plant.  At  noon  on  June  27  a  third  plant  was  added  to  the  series. 
If  the  loss  of  the  leaves  made  any  appreciable  difference  in  the  sto- 
matal  movement  there  should  be  no  agreement  in  the  curves  of  the 
three  sets;  if  these  curves  coincide,  the  effect  of  continued  stripping  is 
not  appreciable.  As  the  graphs  show,  no  difference  can  be  detected. 
When  a  large  proportion  of  the  leaves  of  a  plant  are  removed,  there 
can  be  no  doubt  that  stomatal  movement  in  the  remaining  leaves  will 
not  be  normal,  but  this  was  avoided  by  removing  comparatively  few 
leaves  from  each  plant. 

In  the  following  descriptions  and  graphs,  stomatal  apertures  are 
expressed  in  percentages  of  the  maximum.  This  checks  out  the  effect 
of  variations  in  size  of  stomata  and  readily  allows  comparison  of 
stomatal  movement  in  the  various  plants  investigated.  It  also 
allows  the  relation  of  Ught  and  humidity  to  be  more  clearly  shown 
than  is  possible  in  any  other  manner.  As  it  was  not  possible  to  meas- 
ure fractions  of  0.1  micron,  the  degree  of  opening  is  given  to  the 
nearest  twentieth  of  the  maximum.  In  the  earlier  experiments  the 
diffusion  capacity  n  Va6  was  also  calculated,  but  the  curve  produced 
was  essentially  similar  and  had  no  advantage  not  found  in  the  method 
used. 

Acknowledgment  is  due  Dr.  P.  J.  O'Gara,  of  the  American  Smelt- 
ing and  Refining  Company,  for  aid  and  assistance  throughout  this 
investigation.  As  the  1916  work  was  carried  on  for  the  American 
Smelting  and  Refining  Company,  the  use  of  all  material  and  photo- 
graphs from  that  year  is  by  their  permission.  At  various  periods 
since  that  time,  the  company,  through  Dr.  O'Gara,  has  extended  the 
use  of  its  laboratory  and  given  material  assistance  in  the  continuance 
of  the  work.  Acknowledgment  is  also  due  Dr.  Clements  for  making 
possible  the  further  prosecution  of  the  work,  especially  in  connection 
with  the  effect  of  stomatal  movement  upon  transpiration. 


I.  THE  DAILY  MARCH  OF  STOMATAL  MOVEMENT. 

These  experiments  were  originally  undertaken  to  determine  when 
the  stomata  of  the  more  important  crop-plants  of  the  Great  Salt 
Lake  region  were  open.  Experiment  had  shown  that  usually  five 
times  the  SO 2  concentration  required  to  injure  pUnts  during  the  day 
was  necessary  to  produce  a  like  degree  of  injury  at  night.  This  was 
explained  as  a  result  of  the  condition  of  the  stomata,  but  exact  in- 
formation was  needed  as  to  when  stomata  opened  and  closed  during 
a  24-hour  period.  This  was  especially  desirable,  as  prevention  of 
smelter  injury  to  vegetation  was  at  that  time  based  upon  the  "sea 
captain"  plan  of  smelter  operation,  by  which  the  manager  kept  in- 
formed of  the  approach  of  unfavorable  conditions  when  plants  were 
especially  susceptible  to  SO 2  injury,  and  reduced  operations  within 
the  smelter  accordingly. 

At  the  outset  it  was  thought  probable  that  for  each  species  the 
daily  march  of  stomatal  movement  was  the  same  from  day  to  day 
as  long  as  conditions  were  not  extreme.  Lloyd  (1908  :  108)  states 
that  in  Fouquiera  "the  stomata  open  and  close  rather  slowly  and 
maintain  a  maximum  opening  for  about  3  hours  from  9  a.  m.  to  12 
day.  This  maximum  can  not  be  said  to  be  strictly  constant,  but  the 
differences  are  slight  and  within  the  personal  error  of  observation. 
Stomatal  closure  occurs  in  the  early  afternoon,  advancing  steadily 
until  nightfall.  It  is  difficult  to  correlate  this  with  changes  in  the 
surrounding  media."  Nevertheless,  it  was  felt  that  such  similarity 
of  behavior  on  days  of  unUke  weather  conditions  required  demon- 
stration for  each  species  concerned.  Largely  for  this  reason,  the 
same  plants  were  used  in  successive  series  on  days  of  different  weather 
conditions.  By  this  means  it  was  soon  found  that  similarity  of 
behavior  was  by  no  means  the  rule,  and  that  stomatal  movement 
differed  in  the  same  species  from  day  to  day  in  accordance  with  the 
physical  conditions. 

THE  DAILY  MOVEMENT  IN  ALFALFA. 

The  first  series,  consisting  of  strips  of  epiderm  collected  hourly, 
was  begun  at  9  a.  m.  May  8,  1916,  and  finished  at  5  p.  m.  the  same 
day.  Light,  temperature,  and  humidity  readings  were  made  during 
this  period,  as  well  as  in  all  the  other  series.  The  curve  for  stomatal 
movement  in  the  upper  epiderm  of  alfalfa  follows  that  of  sunlight, 
except  for  the  sharp  dip  commencing  at  noon.  The  curve  for  sun- 
light shows  that  the  day  was  cloudless  and  totally  free  from  haze. 
At  9  a.  m.  both  were  at  60  per  cent  of  maximum;  at  10  a.  m.  they 
reached  90  per  cent;  at  11  a.  m.  they  were  at  99  per  cent;  and  at  12 
noon  both  reached  maximum.    Sunlight  continued  at  maximum  for 

18 


DAILY   MOVEMENT  IN   ALFALFA. 


19 


the  next  hour  and  a  half,  and  was  still  98  per  cent  at  2  p.  m.  In  the 
meantime  the  stomata  started  to  close  and  reached  total  closure  at 
2  p.  m.  At  3  p.  m.  they  had  begun  to  open  again,  and  at  4  p.  m.  were 
80  per  cent  open,  the  maximum  for  the  afternoon  (fig.  3).  This 
closure  at  2  p.  m.  was  very  puzzling.  At  the  time  it  was  believed  that 
some  mistake  had  been  made,  that  inadvertently  a  functionless  leaf 


100 
90 
80 
70 
60 
50 
-4-0 
30 
20 
10 


/ 

\ 

c-- 

^ 

\ 

i 

/ 

\ 

f~ 

1 

\ 

\. 

\ 

\ 

^__ 

B_ 

- 

100 
90 
80 
70 
60 
50 
4-0 
30 
20 
10 


7 

^^rt-. 

J  Sl/^  V 

\ 

/B 

; 

7   .'   '~^ 

..'■•■■ 

9     10     II    NOON  I 


9      10      II    NOON   12      3      4-5 


FiQ.  2. — Series  1,  showing  movement  in  upper  stomata  of  alfalfa  (A),  lower  stomata  of  barley 

(B),  sunlight  (C). 
Fio.  3. — Series  2,  showing  movement  in  upper  stomata  of  alfalfa  (A),  sunlight  (B),  temperature 

(C),  humidity  (D). 

had  been  stripped  at  2  p.  m.  or  a  vial  of  badly  diluted  alcohol  had 
been  used.  No  other  explanation  seemed  probable,  as  the  plant  was 
not  wilted,  and  an  examination  of  the  soil  showed  that  there  was 
sufficient  moisture. 

The  next  day  another  series  was  made  of  the  same  plants  and  for 
the  same  length  of  time.  The  morning  started  very  cloudy,  but  it 
slowly  cleared  during  the  late  forenoon.  However,  in  the  early 
afternoon  a  haze  appeared  which  thickened  until  it  was  rather 
cloudy  again  at  5  p.  m.,  when  the  series  ended.  An  entirely  different 
behavior  was  observed,  and  one  that  had  no  direct  relationship  to 
changes  in  light  and  only  a  superficial  one  to  changes  in  relative 
humidity.  The  stomata  were  60  per  cent  open  at  the  start  of  the 
series,  80  per  cent  at  10  a.  m.,  and  85  per  cent  at  11  a.  m.  At  noon 
they  had  closed  to  70  per  cent,  but  had  opened  sHghtly  the  next 
hour  and  reached  maximum  at  2  p.  m.,  remaining  in  this  condition 
to  the  end  of  the  series  (fig.  2).  The  one  similarity  in  the  two  series 
is  the  mid-day  closure.  However,  this  occurred  2  hours  earlier  in 
the  second  than  in  the  first  and  involved  only  15  per  cent  change  in 


20 


THE   DAILY   MARCH   OF   STOMATAL   MOVEMENT. 


comparison  with  total  closure  found  the  preceding  day.  In  series  1 
maximum  opening  occurred  in  the  forenoon,  and  the  widest  opening 
in  the  afternoon  was  80  per  cent,  while  in  series  2  maximum  opening 
occurred  during  the  afternoon  and  only  85  per  cent  was  reached  in 
the  forenoon.  The  conclusion  could  not  be  escaped  that  stomatal 
movement  was  not  uniform  each  day  and  that  weather  conditions 
probably  caused  great  variation  in  the  movement  from  day  to  day. 
It  seemed  significant  that  the  stomata  did  not  open  fully  during  the 
cloudy  morning  of  May  9,  and  not  until  an  hour  after  the  maximum 
of  sunlight  had  occurred. 

Another  series  was  made  of  the  same  plants  on  May  12  in  order  to 
discover  whether  the  mid-day  drop  in  the  curve  was  due  to  the  failure 
of  the  stripping  method  or  was  an  actual  occurrence.    The  day  was 

100 

90 
80 

70 
60 
50 
40 
30 
20 
10 


n  r^ 

_     s 

^'^4      \ 

tjA.-9^z^^ 

-sts  y-^ 

7i\3  4 

iL^    ^7 

~^— '         \i 

T 

8      9      10      II    NOON    1 


3      A      5 


FiQ.  4. — Series  3,  showing  movement  in  upper 
stomata  of  alfalfa  (A),  sunlight  (B), 
temperature  (C). 

one  of  passing  clouds,  and  a  certain  amount  of  haze  prevented  the 
light  from  reaching  maximum  at  any  time.  A  severe  frost  on  the 
10th  had  injured  the  vegetation  to  some  extent,  but  a  preliminary 
examination  showed  that  the  stomata  of  the  alfalfa  plants  were 
functioning  in  spite  of  the  low  temperatures.  The  upper  stomata 
were  40  per  cent  open  when  the  series  started  and  opened  gradually 
to  the  morning  maximum  of  80  per  cent  at  11  a.  m.  Again  mid-day 
closure  was  found,  the  stomata  closing  to  70  per  cent  at  noon  and  to  35 
per  cent  at  1  p.  m.,  reaching  a  minimum  of  20  per  cent  at  2  p.  m.  At 
3  p.  m.  they  had  opened  to  55  per  cent  and  to  65  per  cent  at  4  p.  m., 
in  which  condition  they  remained  until  the  end  of  the  series  (fig.  4). 
At  9  a.  m.,  11a.  m.,  2  p.  m.,  and  4  p.  m.  a  direct  examination  of  the 


DAILY   MOVEMENT   IN   ALFALFA. 


21 


leaves  on  the  plant  showed  the  stomatal  apertures  to  check  with  those 
found  in  the  strips  taken  at  the  same  time.  This  left  no  doubt  as 
to  the  actual  occurrence  of  the  mid-day  closure.  Moreover,  since 
this  curve  does  not  resemble  either  of  the  previous  curves,  it  adds  to 
the  evidence  that,  in  alfalfa  at  least,  the  course  of  stomatil  opening 
varies  greatly  from  day  to  day. 

On  May  23,  1916,  another  series  was  made  with  these  same  plants, 
starting  at  10  p.  m.  and  ending  at  8  a.  m.  the  next  day,  while  on 
May  29,  1916,  a  complementary  series  was  begun  at  8  a.  m.  and 
ended  at  10  p.  m.,  thus  furnishing  a  composite  24-hour  series.  It 
was  hoped  to  find  from  the  first  whether  the  stomata  remained 
closed  all  night,  and  at  what  time  they  opened  in  the  morning. 
The  night  of  May  23  was  cold,  clear,  and  rather  windy.  The  hu- 
midity was  not  high,  averaging  only  45  per  cent.  The  day  of  May 
29  was  clear,  sunny,  and  warm,  reaching  83.5°F.  at  3  p.  m.,  but 


MAY23f«- 


^.- 

^■' 

.... 

"/ 

\- 

-.. 

-.. 

c. 

./ 

1 

'\ 

b" 

'' 

'"/i 

"■ 

// 

1 

1 

// 

^ 

^ 

\ 

^ 

/' 

'B 

toor 

90 
80 


fz     ::3iS     V 

A  -v^ 

:-.    :ti:i^^  : 

~'iF--   wf 

I      ^^ii-  t^^ 

—  ^=--z:-  50 


23A567B  9      10     11  NOON  I       23456789      10 


Fig.  5. — Composite  series  4-5,  showing  movement  in  upper  stomata  of  alfalfa 
(A),  sunlight  (B),  temperature  (C),  humidity  (D). 

after  sunset  the  temperature  dropped  quickly  to  58°  F.  at  10  p.  m. 
The  stomata  were  closed  at  10  p.  m.  when  series  4  started  and  re- 
mained closed  until  midnight.  At  1  a.  m.  they  were  5  per  cent  open; 
at  2  a.  m.  they  were  10  per  cent  open;  but  they  closed  again  the 
following  hour.  The  sun  appeared  at  5^  20™  a.  m.  and  no  further 
opening  was  observed  before  this  time.  At  6  a.  m.  they  had  opened 
to  10  per  cent,  at  7  a.  m.  to  30  per  cent,  and  at  8  a.  m.,  when  this 
series  closed,  they  were  half  open.  At  the  beginning  of  series  5, 
on  May  29,  they  had  opened  to  60  per  cent  at  9  a.  m.  and  con- 
tinued opening  uniformly  to  maximum  at  11  a.  m.,  in  which  condition 
they  remained  until  1  p.  m.  At  2  p.  m.  they  were  closed,  a  change 
of  100  per  cent  occurring  within  the  hour.  At  3  p.  m.  they  had 
opened  to  80  per  cent  and,  except  for  a  slight  closure  at  4  p.  m.,  re- 
mained in  this  condition  until  5  p.  m.  They  then  started  to  close 
gradually  for  the  day,  the  process  being  gradual  and  rather  uniform 
and  not  completed  until  after  9  p.  m.  (fig.  5). 


22  THE   DAILY   MARCH   OF   STOMATAL   MOVEMENT. 

Mid-day  closure  had  now  come  to  be  expected,  but  the  speed  with 
which  the  stomita  closed  and  opened  was  startling,  as  none  of  the 
preceding  series  or  my  other  investigations  had  given  any  hint  of  it. 
Another  surprise  was  the  night  opening  at  1  and  2  a.  m.  in  series  4.  If 
the  stomatal  mechanism  depends  upon  light  alone  for  its  action, 
neither  night  opening  nor  day  closure  should  occur.  The  only  con- 
clusion possible  was  that  some  other  factor  than  Ught  ilso  played 
an  important  role  in  the  behavior  of  the  stomata. 

Some  Ught  is  thrown  upon  this  point  by  series  26,  made  at  the 
University  of  Minnesoti  greenhouse  at  Minneapolis,  Minnesota. 
These  plants  were  not  growing  in  the  field,  but  had  been  trans- 
pUnted  into  pots  in  the  greenhouse  while  dormant  during  the  early 
winter.  On  May  1,  1917,  they  were  moved  out  of  doors  to  i  shel- 
tered corner.  The  series  was  started  at  4  a.  m.  on  Miy  5  and  con- 
cluded at  3  a.  m.  the  next  day.  On  the  whole,  conditions  for  growth 
were  very  good.  The  humidity  was  rather  high,  averaging  41  per 
cent  during  the  day  and  72  per  cent  during  the  night.  The  sunlight 
was  almost  normal,  but  the  temperature  was  at  times  unfavorable. 
The  stomati  began  to  open  before  6  a.  m.  and  continued  very  slowly 
and  uniformly  to  the  maximum  at  noon.  They  remained  open  until 
3  p.  m.  and  then  closed  just  as  slowly  and  uniformly,  closure  being 
completed  between  8  and  9  p.  m.  There  was  no  night  opening  (fig. 
6).  Such  behavior  of  the  stomata  in  the  upper  epiderm  of  alfalfa  is 
probably  typical  of  the  movement  under  favorable  conditions,  save 
in  one  respect.  The  low  morning  temper  xtures  had  an  inhibiting 
effect  upon  the  stomita,  and  caused  the  very  gradual  opening  which 
occurred  in  the  forenoon.  The  absence  of  both  day  closure  and  night 
opening  seemed  to  indicate  that  the  two  were  related  in  some  m inner. 
The  following  series  strengthens  this  hypothesis. 

Series  10,  beginning  June  8,  1916,  at  4  x.  m.  and  ending  at  4  a.  m. 
June  9,  wis  the  first  24-hour  series  made.  It  included,  among  others, 
sets  of  upper  and  lower  epiderm  from  the  leaves  of  alfalfa  and  also 
the  stem  epiderm.  The  day  was  cle  ir  and  warm,  the  sunlight  nearly 
normal,  and  the  temperature  88°  F.  at  2  p.  m.  The  relative  humidity 
reached  74  per  cent  at  night  and  dropped  to  13  per  cent  in  the 
afternoon,  an  average  of  65  per  cent  for  most  of  the  night  and  20 
per  cent  for  the  greater  part  of  the  day.  Conditions  were  typical 
of  the  usual  clear  warm  summery  day  in  the  region  (fig.  7).  At 
the  start  of  the  series  the  upper  stomata  were  20  per  cent  open 
and  the  lower  15  per  cent.  At  5  i.  m.  the  upper  had  opened  to  90 
per  cent  and  the  lower  to  60  per  cent.  At  6  a.  m.  the  upper  were 
wide  open.  The  lower  stomata  remained  at  60  per  cent  until  7  a.  m., 
after  which  they  gradually  closed,  the  process  being  completed  by 
noon.  In  the  meantime,  the  upper  stomata  closed  to  50  per  cent  at 
7  a.  m.  and  to  40  per  cent  at  8  a.  m.,  and  they  opened  to  60  per  cent 


DAILY   MOVEMENT   IN   ALFALFA. 


23 


at  9  a.  m.  They  remained  in  this  condition  an  hour  before  beginning 
to  close,  and  became  completely  closed  at  2  p.  m.  At  3  p.  m.  they 
had  opened  30  per  cent  and  were  still  in  this  condition  at  4  p.  m. 
They  gradually  closed  to  10  per  cent  at  6  p.  m.,  remained  stationary 
for  two  hours,  and  closed  completely  by  9  p.  m.  The  lower  stomata 
remained  closed  from  noon  until  5  p.  m.,  after  which  they  opened 
slowly  to  10  per  cent  at  7  p.  m.  At  8  p.  m.  they  again  closed,  to  remain 
so  until  9  p.  m.  By  10  p.  m.  the  stomata  of  both  surfaces  opened 
5  per  cent,  the  upper  remaining  in  this  condition  to  the  next  hour, 
while  the  lower  opened  to  15  per  cent.  At  midnight  the  lower  were 
closed,  but  the  upper  commenced  opening  again,  reaching  15  per 
cent  at  1  a.  m.,  after  which  they  closed  slowly  once  more,  this  being 


100 

~ 

7 

"T" 

"" 

V 

n 

•7 

\ 

u„ 

/ 

v 

\ 

/ 

•B 

^ 

f< 

'. 

\ 

/ 

•• 

/■ 

' 

/ 

'.' 

\^ 

^ 

/.. 

■ 

\ 

4.      5      6      7      8      9      to     11  NOON  t       2       3AB6789tOI«MT.    t       234. 

Fig.  6. — Series  26,  showing  movement  in  upper  Btomata  of  alfalfa   (A), 
Bunlight  (B). 

/ 

N 

y 

^ 

.. 

-, 

./ 

^ 

\ 

••. 

c 

. 

/ 

k^ 

/■ 

•\'' 

/ 

V" 

■• 

.   / 

' 

' 

X 

\ 

' 

/  \ 

\ 

Jr 

w 

1 

\ 

v- 

K 

^' 

1 

"- 

-^ 

X 

-■ 

^ 

_, 

^'' 

\ 

; 

V 

Fig.  7. — Series  10,  weather  data  for  Jime  8-9,   1916,  sunlight  (A), 
humidity  (B),  temperature  (C). 

completed  by  3  a.  m.  The  lower  stomata  continued  closed  from 
midnight  to  3  a.  m.  After  this  hour  the  stomata  of  both  surfaces 
opened,  reaching  the  same  degree  of  opening  at  4  a.  m.,  as  on  the 
preceding  day.  The  stomata  in  the  epiderm  of  the  stems  were 
somewhat  longer  and  narrower  than  those  of  the  upper  epiderm  of 
the  leaves.  They  seemed  to  run  lengthwise  on  the  stem  in  all  cases. 
Their  behavior  agreed  with  that  of  the  upper  stomata,  except  that 
maximum  morning  opening  continued  until  8  a.  m.  when  they  closed 


24 


THE   DAILY   MARCH   OF   STOMATAL   MOVEMENT. 


gradually  to  60  per  cent  at  10  a.  m.  and  for  a  time  again  showed 
the  same  movement  as  did  the  upper  stomata.  At  3  p.  m.,  however, 
they  continued  to  open,  reiching  50  per  cent  at  4  p.  m.,  while  the 
upper  stomata  remained  at  30  per  cent.  From  this  time  they  closed 
slowly  and  uniformly,  coinciding  at  8  p.  m.  with  the  10  per  cent 
opening  found  in  the  upper  stomata  (fig.  8).  From  this  time  to  the 
end  of  the  series  there  was  no  appreciable  difference  between  the 
upper  and  the  stem  stomata. 


7 

, 

i 

\ 

". 

/ 

\ 

'. 

70 

/ 

\ 

'. 

CO 

/, 

^ 

\ 

/ 

\ 

ID 

// 

\ 

^^ 

\ 

c 

//' 

\ 

^ 

^! 

''' 

\ 

\ 

/ 

\ 

\ 

\ 

/ 

\ 

'. 

, 

\ 

^ 

^ 

/ 

N^ 

k 

/ 

"'' 

N 

^. 

-^ 

dL 

-< 

^sl/'l 

9      10     II   NOON  I 


6      7      8      9      10     II    MT 


FiQ.  8. — Series  10,  showing  movement  of  lower  (A),  upper  (B),  and  stem 
stomata  (C)  of  alfalfa. 

An  examination  of  the  previous  series  showed  that  the  movements 
of  the  stomata  in  the  upper  and  lower  epiderms  of  the  same  leaf  did 
not  necessarily  have  any  relation  to  each  other.  In  series  10  the 
general  differences  in  behavior  are  quite  clearly  shown.  The  stomata 
in  the  lower  surface  neither  opened  as  widely  nor  stayed  open  as 
long  as  the  stomata  of  the  upper  epiderm.  One  reason  for  this  is  that 
the  lower  surface  of  the  leaf  receives  much  less  light  than  the  upper, 
but  it  seems  largely  to  be  a  matter  of  water-supply  to  the  guard-cells. 
The  slower  opening  in  the  morning  and  earlier  closing  in  the  evening 
typical  of  the  lower  surface  are  due  to  the  difference  in  light,  but  this 
is  not  true  of  the  prolonged  mid-day  closure  and  slight  afternoon 
opening  in  the  lower  stomata.  Experiments  made  by  turning  the 
leaf  over  and  keeping  it  upside  down  during  the  day  seem  to  bear 
this  out,  but  have  not  been  conclusive  at  all  times.  In  barley  and 
other  plants  with  leaves  that  may  have  either  side  up,  the  stooiata 
on  both  surfaces  behaved  ahke  for  the  most  part.  In  the  cases  where 
they  did  not,  the  difference  was  clearly  due  to  difference  in  lighting, 
and  had  no  relation  to  the  side  on  which  the  stomata  were  found. 

Another  point  of  importance  was  the  behavior  of  individual  stomata. 
Direct  observation  showed  that  individual  stomata  may  become 
functionless  from  one  cause  or  another  and  remain  permanently 
closed.  Some  seemed  to  have  the  ability  to  open  much  more  widely 
than  others.  Moreover,  those  that  opened  very  widely  also  opened 
earlier,  reached  maximum  opening  sooner  than  the  others,  and  re- 


DAILY   MOVEMENT   IN   ALFALFA.  25 

mained  widely  open  for  a  somewhat  longer  time.  Complete  closure 
occurred  at  approximately  the  same  time,  and  hence  these  more 
active  stomata  closed  more  rapidly  than  the  others,  as  well  as  opening 
more  widely.  In  most  sets  of  epiderm  examined  were  found  com- 
plete series  of  stomata,  from  these  very  functional  ones  to  others 
which  seemed  entirely  functionless.  By  keeping  a  leaf  under  the 
microscope,  held  on  a  slide  without  a  cover-glass,  and  shutting  off 
the  light  between  periods  of  observation,  this  individual  variation  in 
the  action  of  the  stomata  could  be  followed  in  detail.  Naturally, 
the  behavior  of  the  stomata  of  a  leaf  treated  in  this  manner  is  not 
like  the  behavior  of  the  stomata  on  the  other  leaves  of  the  plant. 
Still  it  may  be  safely  assumed  that  when  there  is  a  more  or  less  indi- 
vidual reaction  by  each  stoma  to  rather  unnatural  conditions,  there 
is  also  similar  variation  under  more  natural  conditions.  This  is  the 
only  assumption  that  will  explain  the  differences  in  the  degree  of 
opening  found  in  the  various  stomata  of  a  strip  or  at  the  moment 
of  observation  in  a  living  leaf.  However,  most  of  the  stomata  act 
alike;  at  least  80  of  each  100  in  alfalfa  are  alike  within  the  accuracy 
of  measurement,  and  the  greater  part  of  the  remaining  20  are  suffi- 
ciently near  the  average,  so  that  they  have  no  effect.  Approxi- 
mately 2  per  cent  of  all  the  stomata  in  an  alfalfa  leaf  are  function- 
less,  because  of  incomplete  development  or  a  more  obscure  cause,  and 
3  per  cent  are  superfunctional,  opening  often  to  nearly  twice  the 
normal  maximum  and  usually  changing  the  degree  of  opening  with 
remarkable  speed.  These  erratic  ones  counterbalance  each  other,  and 
have  no  real  effect  upon  the  opening  found  in  a  leaf,  except  during 
a  period  of  stress.  In  cereals,  however,  this  variation  is  important 
at  all  times. 

From  June  8  to  September  1,  1916,  16  additional  series  were 
made,  each  continuing  over  a  period  of  24  hours  or  more,  while  1 
was  68  hours  in  length;  12  of  these  series  contained  a  set  of  alfalfa 
strips,  largely  as  a  basis  for  comparison  with  the  stomata  of  other 
plants.  Figure  9  shows  the  curves  of  the  stomatal  movement  in  the 
upper  epiderm  of  alfalfa,  plotted  so  as  not  to  intersect  each  other. 
In  addition  to  the  5  already  given,  6  have  been  selected  to  show  the 
progressive  changes  in  movement  as  the  soil  became  drier,  the  weather 
hotter,  and  the  evaporating  power  of  the  air  increased.  The  last  two 
curves  are  of  special  interest.     Edith  B.  Shreve  (1916: 114)  states: 

"Under  typical  conditions  the  stomata  [of  Opuntia  versicolor]  begin  to  close 
soon  after  sunrise  and  they  appear  to  be  completely  closed  by  noon.  They 
begin  to  open  at  5  to  6  o'clock  in  the  evening  and  continue  to  open  through- 
out the  night,  the  maximum  size  occurring  between  3  and  6  a.  m." 

This  statement  applies  almost  word  for  word  to  the  behavior  of 
alfalfa  stomata  under  conditions  of  low  water-content,  high  temper- 
ture,  continuous  brilliant  sunshine,  and  very  dry  air;  in  other  words, 


26 


THE   DAILY   MARCH   OF   STOMATAL   MOVEMENT. 


J         ^    ^ 

t      J      ^     ^ 

-J              -V                  -5          \ 

t      t          5  J 

A     ^       7     -^     V 

L     Z^v  t       %  X 

1    Jl  5^         3  J 

7     // JS              V  V- 

t  J    z:  r     ^-3  1 

^^     tz  %  1        ^^            ^Zl 

n    %:t 

7Z     iptzs                   ^"-^ 

z:l7  Tiii                   ^»' 

L_             il i                                                                       S«neaS 

7       KSZL 

Zi_    ^L         12       it 

t    Z           M       3 

1    \  /                 /         ^ 

I   AL              JE           V 

I  J^l  z     it         5 

/    /\4(    \                       \ 

_LZ     it        ^     it                   ^::^        ^^Sc*P»ih 

rX     ^     J.  \                     "^•*"« 

2     Ztl        SI      V.     ^      ,^^ 

Z-    y^^4           ^L             S^-^    \.^^K 

,—        L-i           ^       ^                                                       VL        ^        ^                    Seneara 

"sZI          ^                        .-'^          ^v.    7^" 

i7v-^ 

M  xt 

^-^  ^I                                z^^ 

ZI      1                                Z7S^s^»» 

A3                       -,^   2  L^tZl 

L.   L^  I        ^<             7E  tl 

t-j   ITJ   ^      ^          Z          L 

II^ZIIJ                            Z 

^A2    3                     7   S^    Z           7'Z 

^U                                 ^^^ '^       ^^                            '-5s^„ 

SI   Z   t                                    ^^      '*^' 

J.          \-\          ^^                                                                            <^                               S^MJl 

^    Z:    EJ    7"-^       ^v,    ^-^            7"^ 

I                                            / 

4                                       -.^ 

4                                     Z 

^v                    Z 

^-3I^_^=.    ^-=.Z 

3-4.     5678©     to    II  NOON  I      2     3     4     5     6     7 


9     10    II  MT.    I     2    3 


Fxo.  9. — Movement  in  upper  stomata  of  alfalfa,  showing  change  from  day  opening  and  night 
closure  to  night  opening  and  day  closure,  as  evaporation  became  more  intense. 


DAILY   MOVEMENT   IN    POTATO.  27 

when  conditions  approach  those  under  which  this  Opuntia  grows. 
The  latter  has  adapted  itself  to  such  conditions,  and  hence  such  move- 
ment is  typical  of  its  stomata.  Alfalfa,  under  favorable  conditions, 
has  a  stomatal  curve  typical  of  the  ordinary  mesophyte,  but  it  can 
adjust  itself  to  changing  conditions  until  its  stomatal  curve  is  like 
that  of  an  extreme  xerophyte. 

This  naturally  raised  a  question  as  to  the  factors  concerned.  The 
accepted  view  is  that  light  is  the  all-important  factor  in  stomatal 
movement.  Where  the  plant  is  not  subjected  to  extreme  conditions 
this  is  largely  true,  as  many  investigators  have  shown.  However, 
night  opening  and  day  closure  of  the  stomata  can  not  possibly  be 
attributed  to  light,  but  must  be  due  to  some  factor  or  group  of  factors 
which  counteract  the  effect  of  sunlight.  Moreover,  night  opening 
and  day  closure  must  be  related,  since  in  every  series  an  increase  in 
one  was  accompanied  by  an  increase  in  the  other.  The  most  prom- 
ising explanation  seems  to  be  that  both  result  from  the  same  factor 
changes,  and  a  study  of  the  data  for  the  group  of  series  pointed  to 
two  factors,  in  which  changes  were  parallel  to  those  in  the  daily 
movement  of  the  stomata  of  alfalfa.  These  two  factors,  evapora- 
tion and  water-content,  act  upon  the  plant  singly  or  together  to 
produce  a  water  shortage  or  "incipient  wilting,"  which  brings  about 
closure  of  the  stomata  during  the  day.  Changes  of  no  other  factor 
could  be  correlated  with  day  closure  of  the  stomata,  but  in  all  series 
increased  evaporation  or  decreased  water-content,  or  the  combina- 
tion of  the  two,  increased  day  closure  and  night  opening. 

THE  DAILY  MOVEMENT  IN  POTATO. 

A  set  of  upper  and  lower  epiderm  from  the  leaves  of  potato  was 
included  in  series  10,  June  8  and  9,  1916,  in  order  to  compare  the 
stomatal  movement  of  this  crop-plant  with  that  of  alfalfa.  The 
potato  plants  were  still  very  young  and  it  was  difficult  toward  the 
end  of  the  series  to  find  leaves  sufficiently  mature  to  strip.  The 
soil  conditions  were  not  the  same  for  the  two  plots,  as  the  potato 
plot  had  a  water-content  of  29  per  cent,  which  was  very  high  for 
the  type  of  soil,  while  the  alfalfa  plot  had  but  16  per  cent  at  2  feet. 
However,  the  roots  of  the  alfalfa  plants  undoubtedly  reached  to  the 
moister  soil  just  above  the  water-table,  which  was  at  that  time  only 
6  feet  below  the  soil  surface.  The  weather  conditions  were  the  same 
for  both  plots  (fig.  7). 

The  stomatal  behavior  in  potato  was  entirely  different  from  that 
found  in  alfalfa.  At  the  start  of  the  series  the  stomata  of  both 
surfaces  of  the  leaves  were  wide  open  and  remained  open  throughout 
the  day.  After  sundown  the  lower  stomata  were  closed  for  3  hours 
from  9  p.  m.  until  after  11  p.  m.  At  midnight  they  were  half  open 
and  reached  the  maximum  again  at  1  a.  m    The  upper  closed  to  50 


28 


THE    DAILY   MARCH    OF   STOMATAL   MOVEMENT. 


per  cent  at  9  p.  m.,  but  opened  at  once  and  were  wide  open  the  fol- 
lowing hour.  Hence,  as  in  alfalfa,  the  behavior  of  the  stomata  was 
different  in  the  upper  and  lower  surfaces  of  the  leaves  (fig.  10).  The 
striking  difference  between  the  two  species  lies  in  the  widely  open 
stomata  of  potato  throughout  the  day  and  most  of  the  night.  This 
behavior  indicates  that  light  does  not  produce  opening  of  stomata 
in  this  species.  Moreover,  though  the  greatest  difference  in  envi- 
ronment of  the  upper  and  lower  stomata  is  one  of  light  intensity, 
this  can  hardly  explain  the  behavior  of  the  lower  stomata,  since  these 
did  not  close  until  darkness  set  in.  If  lack  of  light  caused  closure, 
the  upper  stomata  should  have  closed  to  even  a  greater  degree,  since 
these  were  exposed  to  a  greater  change  in  lighting. 


\_W / 

—  —  Q 


10     1 1  NOON 


Fio.  10.- 


-Series  10,  showing  movement  in  upper  (A)  and  lower  (B)  stomata 
of  potato. 


The  tendency  of  potato  stomata  to  remain  continually  open  is 
again  shown  in  series  12,  started  at  1  p.  m.  on  June  21  and  continuing 
to  5  p.  m.,  June  22,  1916.  In  this  case  the  series  was  begun  on  a 
rainy  day  and  ended  on  a  clear  day.  The  light  intensity  was  not  over 
6  per  cent  on  the  afternoon  of  June  21  until  6  p.  m.,  when  the  stopping 
of  the  rain  caused  a  rise  to  13  per  cent.  The  temperature  was  also 
low,  averaging  46°  F.  on  this  afternoon,  but  rising  to  53°  F.  after 
6  p.  m.  The  humidity  averaged  85  per  cent  during  the  afternoon 
and  night.  The  next  day  was  clear,  except  for  a  large  passing 
cloud  at  10  and  11  a.  m.,  although  some  haze  due  to  the  rain  of  the 
previous  day  prevented  the  sunlight  from  reaching  a  maximum.  The 
temperature  was  40°  F.  just  before  daylight,  but  rose  quickly  after 
sunrise,  averaging  62°  F.  during  the  forenoon.  The  humidity  dropped 
to  an  average  of  40  per  cent  during  the  forenoon,  which,  however, 
was  high  for  the  region  (fig.  11).  The  behavior  of  the  stomata  was 
essentially  identical  with  that  found  in  series  10.  Comparison  with 
the  behavior  found  in  alfalfa  stomata  shows  some  interesting  facts. 

The  low  light  intensity  during  the  rainy  afternoon  of  June  21 
caused  the  alfalfa  stomata  to  be  almost  closed.  At  6  p.  m.,  with  the 
increase  of  Hght,  they  opened  to  80  per  cent,  remaining  in  this  con- 


DAILY   MOVEMENT   IN   POTATO. 


29 


dition  until  7  p.  m.  They  then  closed  gradually  until  9  p.  m.  They 
remained  closed  until  4  a.  m.,  when  they  showed  slight  opening. 
At  5  a.  m.  they  were  20  per  cent  open  and  at  6  a.  m.  half  open.  At 
7  a.  m.  this  had  increased  to  90  per  cent  and  at  8  a.  m.  to  maximum. 
The  stomata  remained  in  this  condition  for  an  hour,  but  at  10  a.  m. 
they  had  closed  to  60  per  cent,  at  11  a.  m.  to  50  per  cent,  and  at 


trace 

^ 

(trace 

JULY  26 

JULY  £7, 

816 

HA.I 

1 

L 

/ 

00 

^ 

^ 

-" 

\ 

B  , 

/ 

-^ 

/ 

•-. 

~^.> 

..._. 

\- 

. 



"'/ 

r 

■••c 

~~ 



— 

■^ 

•••, 

1 

■■■•■ 

■•••... 

1 

\ 

■••■ 

A 



/I'l 

/ 

\ 

l\ 

\ 

^_ 

V 

u 

-^ 

10     II    NOON  I       2      3      4      S       6 


7     0     9     10    tr 


Fio.  11.— Series  12,  weather  data  for  June  21-22,  1910;  sunlight  (A),  humidity  (B), 
temperature  (C). 

noon  they  opened  again  to  maximum.  This  forenoon  closure  is 
coincident  with  decrease  in  light  caused  by  the  passing  cloud.  The 
stomata  remained  in  this  condition  3  hours,  closing  to  70  per  cent 
at  4  p.  m.  and  to  45  per  cent  at  5  p.  m.,  when  the  series  ended  (fig.  12). 


^ 

■^ 

"~ 

A 

"7 

n- 

— 

\  ■■ 

/ 

',' 

( 

\. 

\ 

/ 

^ 

1 

( 

\ 

/ 

\ 

1 

\ 

T 

) 

\ 

' 

'v^ 

1 

\ 

( 

-\ 

C/' 

1 

^, 

/ 

1 

/ 

\ 

/ 

\ 

/ 

^- 

-'' 

_ 

2     3     4-      5     6      7      8      9     «3    H     MT. 


23456769      10 


N00t<  I      2      3     *      S 


FiQ.  12. — Series  12,  showing  movement  in  upper  (A)  and  lower  (B)  stomata  of  potato, 
and  upper  stomata  of  alfalfa  (C). 

The  relation  of  stomatal  movement  in  alfalfa  to  changes  in  light 
is  distinct  in  this  series.  Evaporation  was  low  and  the  water-content 
high  as  results  of  the  rain,  which  explains  why  movement  in  the 
stomata  of  alfalfa  was  related  to  light  alone.  But  neither  the  low 
light  intensity  on  the  afternoon  of  June  21  nor  the  darkness  of 
night  caused  any  closure  whatever  in  the  upper  stomata  of  potato. 
The  lower  closed  for  an  hour  at  9  p.  m.,  this  being  the  only  closure 
observed.  The  conclusion  is  inescapable  that  light  has  no  direct 
efifect  upon  stomatal  movement  in  the  potato. 


30 


THE   DAILY   MARCH   OF   STOMATAL   MOVEMENT. 


The  tendency  of  potato  stomata  to  remain  constantly  open  may 
be  ascribed  to  two  causes,  namely,  to  a  large  amount  of  leaf -water 
in  the  thick  and  rather  fleshy  leaves  and  to  efficient  roots  and  high 
root-pressure.  To  show  the  difference  in  root-pressure,  two  mercury 
manometers  were  attached  to  potted  plants  of  alfalfa  and  potato 
and  the  plants  were  heavily  watered.  The  potato  reached  its  max- 
imum in  5  hours,  while  alfalfa  took  12  hours,  and  the  maximum 
for  potato  was  three  times  that  of  alfalfa.  When  tried  with  plants 
growing  in  the  open,  the  same  experiment  failed,  as  both  plants 
showed  negative  pressure,  probably  due  to  inability  to  raise  the 
water-content  sufficiently  during  the  experiment.  While  too  much 
reliance  can  not  be  placed  upon  this  experiment,  it  is  believed  that 
it  is  fairly  indicative  of  the  relative  efficiency  of  the  roots  of  the  two 
plants.  Although  conditions  during  the  two  series  described  were 
very  unlike  in  most  respects,  the  stomata  of  potato  behaved  in 
essentially  the  same  manner.  This  was  due  to  the  high  water-content 
of  the  soil  during  both  experiments.  When  the  water-content  drops 
below  the  amount  necessary  under  given  conditions  of  humidity 
and  temperature,  potato  stomata,  as  well  as  alfalfa  stomata,  react 
to  conserve  water.  When  the  water-content  is  but  little  below  the 
amount  required,  closure  of  the  lower  stomata  occurs  in  the  late 
afternoon,  followed  by  partial  or  total  closure  of  the  upper  stomata. 
As  soon  as  the  leaves  regain  turgor,  the  stomata  again  open  and  stay 
open  the  remainder  of  the  night.  In  all  cases  studied  the  upper 
stomata  remained  open  longest  and  were  the  first  to  reopen  when  the 
lost  leaf -water  was  replaced. 


"1 

-^ 

. 

y~t 

80 

" 

b\ 

/ 

^ 

7 

ff\ 

bs 

^ 

^ 

I::^ 

/ 

■-*- 

-" 

" 

r-^ 

V 

/ 

^ 

y 

50 

'S 

/ 

s 

/ 

— 

Ar 

^ 

V 

/^ 

*\ 

/ 

c 

/ 

\ 

^ 

^ 

; 

\ 

20 
10 

— 

K^^ 

\ 

; 

,[ 

■-^ 

... 

V 

J 

[S 

^ 

/- 

10     II     MX    I 


8     9      10     n    NOON  I 


Fio.  13.— Series  20,   weather  data  for  August  25-26,   1916;  sunlight   (A), 
temperature   (B),   humidity  (C). 

This  is  shown  by  series  20  (fig.  14),  which  was  begun  at  noon  August 
25,  1916,  and  ended  at  1  p.  m.  the  following  day.  On  both  days  the 
light  was  strong,  although  a  certain  amount  of  haze  was  present,  the 
temperatures  high,  and  humidity  low.  The  soil  had  dried  to  a 
water-content  of  15  per  cent,  which  was  not  quite  sufficient,  in  view 
of  the  high  evaporation.     At  noon,  when  the  experiment  started. 


DAILY   MOVEMENT   IN    POTATO. 


31 


the  stomata  of  both  surfaces  of  the  leaves  were  wide  open.  At  1 
p.  m.  the  lower  had  closed  to  30  per  cent,  although  the  upper  were 
still  open.  At  2  p.  m.  the  lower  stomata  were  closed  and  the  upper 
were  but  40  per  cent  open.  By  3  p.  m.  the  stomata  of  both  surfaces 
were  closed  and  so  remained  until  9  p.  m.  when  the  upper  stomata 
showed  a  slight  amount  of  opening.  At  10  p.  m.  the  lower  stomata 
also  started  to  open;  the  upper  had  then  opened  to  30  per  cent  of 
maximum.  At  11  p.  m.  the  upper  stomata  were  60  per  cent  open  and 
the  lower  15  per  cent.  At  midnight  the  upper  stomata  were  wide 
open  and  the  lower  were  40  per  cent  open.  At  1  a.  m.  the  lower 
stomata  were  80  per  cent  open  and  fully  open  at  2  a.  m.  The  stomata 
in  both  surfaces  were  open  until  11  a.  m.,  when  the  lower  stomata 
suddenly  began  to  close.    At  12  noon  the  lower  were  but  10  per  cent 


r 

1 

f 

r 

\ 

\ 

'. 

\- 

\ 

00 

\ 

1 

1 

1 

1 

i  I 

I 

1 

\ 

1 
\ 

'a 

\  ' 

\ 

10 

\ 

1 

\ 

\ 

1 

1 

\ 

\ 

in 

\ 

\ 

1 

\ 

\ 

^ 

/ 

_ 

"^ 

., 

10     II   UOOM  I 


FiQ.  14. — Series  20,  showing  movement  in  upper  (A)  and  lower  (B)  stomata 
of  potato,  plot  7. 

open  and  the  upper  stomata  started  to  close.  At  1  p.  m.,  when  the 
series  ended,  the  lower  epiderm  showed  all  stomata  closed  and  the 
upper  but  15  per  cent  open.  The  stomatal  movement  in  the  upper 
epiderm  of  alfalfa  for  the  same  time  shows  that  potato  is  not  so 
susceptible  to  evaporation  as  alfalfa.  Nevertheless,  the  stomata  of 
potato,  Uke  those  of  alfalfa,  are  influenced  by  these  factors. 

Less  available  water  tends  to  increase  the  day  closure.  When  it 
is  very  much  less,  the  stomata  close  very  early  and  the  entire  plant 
shows  signs  of  wilting.  During  the  night  such  a  plant  will  recover 
and  its  stomata  again  open.  When  the  small  reserve  of  water  gained 
overnight  begins  to  disappear,  the  stomata  again  close  and  the 
plant  assumes  once  more  a  semi-wilted  appearance.  Hence,  as  in 
alfalfa,  the  stomata  of  potato  react  to  excessive  water-loss  by  closure. 
This  cuts  down  the  period  for  the  absorption  of  carbon  dioxide  and 
naturally  the  amount  of  photosynthesis  which  can  occur  in  such  a 
plant,  without  regard  to  other  effects  due  to  water-loss. 

The  stomata  of  potato  show  other  interesting  differences  as  com- 
pared with  those  of  alfalfa.  One  variety,  the  name  of  which  was  un- 
known, had  hypostomatal  leaves.     The  variety  used  in  these  experi- 


32 


THE    DAILY   MARCH   OF   STOMATAL   MOVEMENT. 


ments  (Russet  Burbank)  had  three-twentieths  as  many  stomata  on  the 
upper  surface  as  on  the  lower.  Unlike  alfalfa,  the  lower  stomata  were 
more  active  than  the  upper.  All  the  stomata  of  an  alfalfa  leaf  become 
functional  at  practically  the  same  time.  In  potato,  however,  the 
first  stomata  may  begin  to  function  long  before  the  last  have  formed. 
Two  weeks  have  elapsed  in  most  cases  between  the  time  the  first 
stoma  on  a  leaf  opened  and  the  last  stomata  became  functional. 

The  curves  made  by  the  lower  stomata  of  potato  under  three 
conditions  of  water-content  are  shown  in  figure  15.  The  last  curve 
is  much  the  same  as  that  of  alfalfa  under  similar  conditions,  as  it 


.00 

r- 

■~~ 

"7 

*^ 

r 

■"" 

■"" 

V 

1 

_/ 

*v 

/ 

\ 

\ 

h 

i 

\ 

' 

t 

\ 

50 

\c 

\ 

,'i 

A 

', 

b\ 

Jl 

/ 

1 

/b 

1 

1 

•0 

' 

1 

^-- 

-- 

•^ 

1 

A 

MT.    r      2      3 


Fio.  15. — Movement  in  lower  stomata  of  potato  under  high  water-content 
and  moderate  evaporation  (A),  low  water-content  and  moderate 
evaporation  (B),  low  water-content  and  excessive  evaporation  (C). 

shows  day  closure  and  night  opening.  But  where  alfalfa  under  very 
favorable  conditions  has  stomata  open  all  day  and  closed  at  night, 
potato  has  stomata  open  continuously,  save  for  the  3  hours  following 
sundown.  Then,  as  the  conditions  for  obtaining  and  retaining  water 
become  less  favorable,  this  period  of  closure  begins  earlier,  until 
finally  it  includes  all  of  the  day  except  an  hour  or  two  at  sunrise. 
There  was  in  no  case  the  mid-day  closure  and  afternoon  opening  so 
often  found  in  alfalfa  stomata.  This  may  occur  possibly  in  other 
varieties  of  potato  or  under  other  conditions,  but  was  not  found  in 
this  investigation.  This  would  seem  to  indicate  that  a  larger  pro- 
portion of  the  leaf-water  was  lost  by  the  potato  before  its  stomata 
closed  than  is  the  case  in  alfalfa.  The  slightly  wilted  appearance 
of  the  plants  at  the  times  when  their  stomata  are  closed  would 
confirm  this,  as  there  is  no  sign  of  such  wilting  of  alfalfa  plants  during 
the  midday  closure  of  their  stomata.  The  more  ready  closing  of 
stomata  in  alfalfa  may  in  part  explain  its  greater  drought-resistant 
qualities  as  compared  with  potato.  It  is  obvious  that  a  plant  which 
waits  until  the  last  moment  to  cut  down  water-loss  must  waste 
much  more  than  the  plant  which  is  so  adjusted  as  to  cut  down  this 
loss  at  the  first  signs  of  shortage.  On  the  other  hand,  it  seems 
reasonable  that  when  the  water-supply  is  adequate,  but  evaporation 


DAILY   MOVEMENT   IN    SUGAR-BEET.  33 

is  high,  the  potato  with  its  stomata  open  throughout  the  day  can 
produce  much  more  photosynthate  than  alfalfa,  which  has  the 
stomata  closed  for  a  considerable  portion  of  the  working  period. 

THE  DAILY  MOVEMENT  IN  SUGAR-BEET. 

The  stomata  and  epidermal  cells  of  sugar-beet  are  unique  in  being 
similar  on  both  surfaces  of  the  leaf.  This  is  in  marked  contrast  to 
the  epiderm  of  alfalfa  or  potato  leaves,  where  the  stomata  and  cells 
of  one  surface  are  distinctly  unlike  those  of  the  other.  For  this 
reason,  as  well  as  because  of  its  importance  as  a  crop  plant  in  the 
region  about  the  Great  Salt  Lake,  this  plant  was  included  in  a  number 
of  series.  The  first  of  these  was  No.  11,  started  at  11  a.  m.  June  19, 
1916,  and  ended  at  noon  June  20.  The  weather  conditions  of  these 
two  days  were  characterized  by  increasing  humidity,  high  tempera- 
ture, and  cloudy  nights,  which  culminated  in  a  rain  on  June  21. 
Throughout  this  period  passing  light  clouds  cut  down  the  sunlight, 
but  at  no  time  to  the  point  where  the  stomata  were  affected.  The 
temperature  ranged  from  80°  F.  at  3  p.  m.  June  19  to  55°  F.  just 
before  dawn  on  June  20.  A  dense  layer  of  clouds  formed  during  the 
evening  of  the  19th  and  persisted  until  after  sunrise.  This  condition 
was  responsible  for  the  warm  night  and  resulted  in  the  high  humidity 
of  the  following  day  (fig.  16).  The  light  was  not  as  great  or  the 
temperature  as  high  this  second  day  as  on  the  first. 

At  the  start  of  the  series  the  upper  stomata  had  just  started  to 
close,  while  the  lower  showed  but  30  per  cent  opening.  The  lower 
surface  reached  a  minimum  of  5  per  cent  for  the  afternoon  at  1  p.  m. 
and  remained  in  this  condition  for  an  hour.  The  upper  had  closed 
to  10  per  cent  at  2  p.  m.  At  3  p.  m.  both  surfaces  showed  the  stomata 
opening;  at  4  p.  m.  the  lower  had  opened  to  40  per  cent,  while  the 
upper  had  only  reached  20  per  cent.  The  next  hour  the  lower  had 
opened  to  50  per  cent,  but  the  upper,  increasing  the  rate  of  opening 
very  much,  reached  80  per  cent.  This  was  the  time  of  greatest 
opening  in  either  surface  that  afternoon.  The  following  hour  the 
lower  stomata  were  unchanged,  but  the  upper  had  closed  sUghtly, 
and  then  more  rapidly  until  nearly  all  were  closed  at  10  p.  m.  The 
lower  stomata  closed  to  10  per  cent  at  8  p.  m.  and  then  began  to 
open,  reaching  a  maximum  for  the  night  of  70  per  cent  at  11  p.  m. 
At  this  hour  the  upper  stomata  had  opened  slightly  and  reached 
15  per  cent  at  1  a.  m.  After  11  p.  m.  the  lower  stomata  had  gradually 
closed,  this  change  being  completed  at  3  a.  m.  The  upper  stomata 
reached  the  minimum  of  7  per  cent  at  the  same  time.  The  next  hour, 
although  the  light  of  approaching  dawn  was  very  faint,  the  upper 
stomata  had  opened  60  per  cent  and  were  wide  open  at  5  a.  m.,  about 
20  minutes  before  the  sun  rose  over  the  mountains.  The  lower 
stomata,  however,  were  still  closed  at  4  a.  m.  and  showed  only  5  per 


34 


THE   DAILY   MARCH   OF   STOMATAL   MOVEMENT. 


cent  opening  at  5  a.  m.,  when  the  upper  had  reached  the  maximum. 
The  next  hour  the  lower  opened  swiftly  to  60  per  cent  and  then 
gradually  to  90  per  cent  at  10  a.  m.,  which  was  the  forenoon  maxi- 
mum of  these  stomata.  The  upper  continued  wide  open  until  11 
a.  m.  and  then  closed  to  90  per  cent  at  noon,  when  the  series  ended. 
The  lower  started  to  close  after  10  a.  m.,  reaching  65  per  cent  at 
11  a.  m.  and  35  per  cent  at  noon  (fig.  17). 

The  lower  stomata  opened  2  hours  later  in  the  morning  than  did 
the  upper,  and  closed  2  hours  earHer  in  the  evening.  The  smaller 
amount  of  light  received  by  them  affords  ready  explanation  of  this. 
However,  the  greater  mid-day  closure  and  much  greater  opening 
found  in  these  stomata  at  night  can  not  be  explained  on  the  basis 


JUNE  19  — »k—  JUNE  20 


"V  ^^     i                               /^ 

„-±2-=^^^-  -^                                        t   V 

"   JL        t        ^■-=';-^               ^^.-dE-^ 

z         i    -^     ^'         ii  I    '^ 

z~        i  ,7                ft /^ 

^            .IT    •'^                                                   '^^/ 

IC-..                       ^^ 

12    IRM.  2      3     4       3      6 


9      10     li     12  IAM2     3     4      S      6 


6     9      10    II      12   IPM.  2 


Fig.  16.- 


-Series  11,  weather  data  for  June  20-21,  1916;  sunlight  (A),  humidity  (B), 
temperature  (C). 


100 

/ 

'~~ 

\1 

/ 

^■^ 

•'' 

\ 

1 

i 

■^ 

/ 

/ 

\ 

/ 

\ 

^ 

/ 

\ 

/ 

^ 

\ 

^ 

/ 

\ 

/ 

\ 

\^ 

so 

/' 

\ 

\ 

1 

\B 

/ 

M 

iO 

\ 

/ 

/ 

\ 

\ 

1 

\ 

/ 

20 

\ 

\ 

/ 

^, 

y 

-. 

/ 

\ 

\ 

^ 

^ 

'^^ 

y 

\ 

r^ 

•^ 

S 

/ 

^1 

^ 

J 

^ 

^1 

^■ 

J 

II   NOON  I       23*567e8WIIMT.     I       23-4.96769       10    II    NOON 

Fia.  17. — Series  11,  showing  movement  in  upper  (A)  and  lower  (B)  etomata  of 
sugar-beet. 

of  light  intensity,  in  spite  of  the  fact  that  this  is  the  greatest  differ- 
ence in  the  environment  of  the  two  surfaces.  In  fact,  the  alfalfa 
series  would  indicate  that  the  upper  stomata  should  show  greater 
night  opening  and  day  closure  than  the  lower.  On  the  other  hand, 
the  shaded  soil  in  the  alfalfa  plots  did  not  give  rise  to  convection 
currents  of  great  evaporating  power  striking  the  lower  surfaces  of 
the  leaves,  such  as  rose  from  the  hot,  bare  surface  of  the  cultivated 
soil  of  the  sugar-beet  field. 


DAILY   MOVEMENT   IN    SUGAR-BEET. 


35 


On  July  20,  1916,  thirty  leaves  of  sugar-beet  were  carefully  twisted 
so  as  not  to  injure  them  and  left  clamped  with  their  lower  surfaces 
turned  upward.  In  the  following  discussion  upper  and  lower  sur- 
face are  used  to  describe  the  respective  surfaces  of  the  leaves,  regard- 
less of  the  actual  position  in  which  they  were  placed.  On  July  26, 
1916,  series  16  was  started  at  9  a.  m.  and  continued  until  11  a.  m. 
the  following  day.  Among  the  sets  of  epiderm  collected  were  two 
of  sugar-beet,  one  from  the  upper  and  lower  surfaces  of  normal 
leaves,  and  one  from  the  two  surfaces  of  the  reversed  leaves.  This 
was  to  determine  how  much  of  the  difference  in  the  behavior  of  the 
stomata  in  the  two  surfaces  of  the  leaves  was  due  to  physical  factors 
and  how  much  to  the  internal  structure  of  the  leaves. 

The  weather  conditions  were  rather  favorable  for  growth.  Two 
light  showers  occurred  on  the  afternoon  of  the  26th  at  3^15°"  and 
4^10™  p.  m.  Each  lasted  but  a  minute  or  two,  and  there  was  not 
sufficient  precipitation  to  measure.  They  did  not  reduce  the  Ught 
sufficiently  to  produce  any  change  in  the  behavior  of  the  stomata. 
The  temperature  and  humidity  were  high  on  both  days.     The  26th 


JUNE  22 

wfunii.   tl 

* 

' 

eo 







.B. 



.... 



... 

.... 

fN 

/ 

■*■ 

\ 



\ 

■••, 

N 

\ 

\ 

-\ 

- 

_- 

\ 







C 

'f: 

1 

\ 

4<5 

-- 

— 

— 



'' 

-- 

— 

-^ 

^- 

—. 

^ 

1. 

, 

^ 

U 

V 

\ 

y 

1 

10 

/ 

r^ 

> 

^ 

_ 

^ 

r 

_ 

' 

t 1 

10      II      12    1A.M.  2 


8      9      10      M      12    IRM-2      i      4-     S 


Fio.  18.— Series  16,  weather  data  for  July  26-27,  1916;  sunlight  (A),  temperature  (B),  humidity  (C). 

especially  was  a  still,  hot  day,  oppressive  because  of  the  high  humidity, 
with  clouds  hanging  over  the  mountains  and  the  valley  in  brilliant 
sunshine.  There  were  fewer  clouds  on  the  day  following,  and 
these  disappeared  as  they  drifted  away  from  the  mountains  (fig.  18). 
The  curves  for  the  two  sets  of  sugar-beet  leaves  are  shown  in  figure 
19.  The  upper  epiderm  of  the  reversed  leaves  produced  the  same 
etomatal  curve  as  the  lower  epiderm  of  the  normal  leaves.  The 
lower  epiderm  of  the  reversed  leaves  did  not  show  exactly  the  same 
stomatal  movement  as  in  the  upper  stomata  of  the  normal  leaves, 
but  the  behavior  was  essentially  similar,  demonstrating  that  most 
of  the  difference  between  the  upper  and  lower  stomata  of  the  normal 
leaf  is  the  result  of  a  different  environment.  The  sUghtly  slower 
opening  and  earlier  closing  of  the  lower  stomata  of  the  reversed  leaves 


36 


THE   DAILY   MAECH   OF   STOMATAL   MOVEMENT. 


may  be  attributed  to  a  less  ready  supply  of  water  to  their  guard-cells. 
Reversing  the  leaves  of  alfalfa  did  not  cause  their  stomata  to  show 
the  results  found  in  sugar-beet,  but  the  stomata  of  each  surface  still 
behaved  differently  from  those  of  the  other  surface  of  normal  leaves. 
Earlier  opening  and  later  closing  of  the  lower  stomata  were  often 
induced  by  reversing  the  leaves,  and  at  times  greater  midday  closure 
was  brought  about. 

It  is  thus  evident  that  the  difference  in  stomatal  behavior  of  the 
upper  and  lower  epiderms  of  alfalfa  is  due  to  differences  in  the  struc- 
ture of  the  leaf,  and,  in  particular,  of  the  stomata  and  surrounding 
epidermal  cells.  The  position  of  the  veins  and  the  number  of  cells 
through  which  the  leaf-water  must  pass  before  reaching  the  guard- 
cells  of  the  stomata  of  each  surface  must  have  considerable  influence 
upon  the  functioning  of  the  stomata,  at  least  under  conditions  of 
high  evaporation.  The  air-spaces  in  the  spongy  chlorenchyma  and 
fewer  paths  or  bridges  of  cells  through  these  air-spaces  from  the 
veins  to  the  epidermal  cells  must  play  a  part  in  the  rate  at  which 
water  passes  to  the  lower  epiderm  and  in  the  loss  by  evaporation 


Z                                         12  V 

»t                                             4  ^h 

"t                                             tJ^V 

«I                                     c'^ut 

zX                    1^        U  Xt 

"4               -,           t  ^       7t  X\ 

iX            ^^v      'A      u    ^A 

X                           -fl.      ^          ^^7%          Xl              r 

,!\                k^S   Vv    2    4fc^    /         ^ 

3^           Z/'      sV^^    jfef   t        3^ 

2      3      4.      5      6      7      e      9      10     II   MT.     I       a       3      *       5      6       7      6      9     10     11 


Fig.  19. — Series  16,  showing  effect  on  movement  of  reversing  leaves  of  sugar-beet; 
normal  leaves,  lower  stomata  (A),  upper  stomata  (B);  reversed 
leaves,  lower  stomata  (C),  upper  stomata  (A),  as  in  lower  stomata 
of  normal  leaves. 

during  its  passage.  As  leaf  sections  show,  the  water  on  its  way  to 
lower  and  upper  epiderm  guard-cells  passes  through  the  same  num- 
ber of  cells  in  sugar-beet,  and  the  air-spaces  in  the  sponge  tissue  are 
not  nearly  so  large  or  numerous  as  in  alfalfa  (plates  5  and  6). 

No  series  was  made  of  sugar-beet  under  conditions  where  normal 
stomatal  behavior  could  be  expected,  nor  was  this  found.  However, 
it  can  not  be  doubted  that  since  the  general  behavior  of  the  stomata 
of  this  species  resembles  that  found  in  alfalfa,  sugar-beet  stomata 
would  show  essentially  the  same  behavior  under  conditions  of  low 
evaporation,  medium  temperature,  bright  sunshine,  and  high  water- 
content,  as  found  in  alfalfa,  series  26  (fig.  6).  The  type  of  curve 
shown  in  each  series  is  like  the  type  found  in  alfalfa  of  the  same 
series,  in  that  the  stomata  respond  to  changes  of  physical  factors  in 


DAILY   MOVEMENT   IN    ONION.  37 

essentially  the  same  manner,  although  not  to  the  same  degree.  The 
outstanding  difference  lies  in  the  fact  that  the  stomata  of  sugar- 
beet  are  alike,  while  those  of  alfalfa  differ  on  the  two  surfaces  of 

THE  DAILY  MOVEMENT  IN  ONION. 

As  the  leaves  of  onion  are  essentially  hollow  cylinders,  with  no 
distinct  upper  and  lower  epiderm,  but  one  set  of  epidermal  strips 
was  collected  in  each  major  series  that  included  this  plant.  However, 
one  special  series  6  hours  in  length  was  made  to  find  whether  the 
difference  in  lighting  on  the  different  sides  of  the  leaf  had  any  effect 
upon  the  behavior  of  the  stomata.  In  addition,  a  number  of  iso- 
lated tests  were  made  for  the  same  purpose.  The  results  showed 
that  the  difference  of  lighting  did  cause  variation  in  the  stomata 
affected  at  certain  times,  and  under  certain  conditions  especially. 
On  this  account,  at  each  stripping  during  a  24-hour  series,  three  to 
four  strips  were  collected  from  the  different  sides  of  the  leaf,  so  that 
the  average  degree  of  opening  for  that  leaf  could  be  found. 

There  are  almost  as  many  stomata  in  onion  epiderm  as  unspecial- 
ized  epidermal  cells,  as  the  end  of  each  such  cell  is  usually  separated 
from  the  next  by  a  stoma  (plate  4).  However,  because  of  the  size 
of  these  cells,  there  are  about  the  same  number  of  stomata  to  a 
square  millimeter  of  epiderm  as  in  a  sugar-beet  leaf.  The  stomata 
are  rather  large  and  unusually  simple  in  form  and  mechanism. 

The  general  type  of  behavior  found  in  the  stomata  of  onion  is 
shown  in  series  16,  started  9  a.  m.  July  26,  and  ending  11  a.  m.  July 
27,  1916.  As  usual,  a  set  of  alfalfa  epiderm  was  collected  in  the 
same  series  for  comparison.  Because  of  the  high  humidity,  neither 
the  onion  nor  the  alfalfa  stomata  showed  the  degree  of  day  closure 
that  would  be  expected  in  view  of  the  high  temperature  and  brilliant 
sunshine  (fig.  18).  At  the  outset,  the  onion  stomata  were  70  per 
cent  open  and  those  of  alfalfa  80  per  cent.  At  10  a.  m.  both  species 
had  stomata  80  per  cent  open,  and  these  began  to  close  immediately. 
By  the  following  hour,  alfalfa  showed  complete  closure  and  remained 
closed  until  3  p.  m.  The  stomata  of  onion  closed  much  more  gradu- 
ally, reaching  40  per  cent  at  1  p.  m.,  when  they  opened  as  gradually 
to  the  maximum  at  5  p.  m.  The  alfalfa  stomata  opened  in  the  mean- 
time and  also  reached  maximum  at  this  hour.  At  6  p.  m.  the  onion 
stomata  had  closed  to  40  per  cent,  remaining  in  this  condition  for  an 
hour,  then  to  30  per  cent  at  8  p.  m.  for  an  hour,  finally  closing  to  10  per 
cent  at  10  p.  m.  The  alfalfa  stomata  closed  uniformly  to  10  per  cent 
at  9  p.  m.,  but  showed  no  further  closure  the  following  hour.  At  11 
p.  m.  night  opening  had  set  in  for  both  species,  onion  reaching  15  per 
cent,  remaining  there  for  an  hour  and  then  closing  slowly  and  com- 
pletely by  2  a.  m.  Alfalfa  stomata,  on  the  other  hand,  opened  gradu- 
ally to  a  maximum  of  50  per  cent  at  1  a.  m.  and  then  closed  slowly 


38 


THE   DAILY   MARCH   OF   STOMATAL   MOVEMENT. 


and  completely  by  3  a.  m.  After  3  a.  m.  the  onion  stomata  began  to 
open  very  slowly  to  20  per  cent  at  5  a.  m.  As  a  result  of  sunrise, 
however,  they  opened  within  the  next  hour  to  an  unstable  maximum 
of  80  per  cent  for  the  morning.  This  maximum  continued  with 
several  fluctuations  until  11  a.  m.  when  the  stomata  closed  to  60 
per  cent,  as  in  the  preceding  day.  In  alfalfa  the  stomata  were 
closed  until  after  4  a.  m.,  when  they  began  to  open  slowly,  reaching 
10  per  cent  at  5  a.  m.,  and  then  more  rapidly  to  80  per  cent  at  8  a.  m. 
They  continued  in  this  condition  for  an  hour,  and  then  closed  to  60 
per  cent  at  10  a.  m.     By  11  a.  m.  they  had  closed  completely. 


/ 

i\ 

/; 

\\ 

/ 

\ 

/ 

,' 

, 

\ 

/ 

\ 

V 

\. 

'A 

/ 

' 

\ 

\ 

/ 

\ 

\ 

\ 

i 

/ 

^ 

1 

1 

\ 

J 

IB 

\ 

/ 

V 

I 

\ 

» 

1 

\ 

/' 

\ 

1 

\ 

■»o 

1 

V 

/ 

\ 

,' 

\ 

1 

/ 

\ 

^ 

— 

\ 

/ 

/ 

^ 

i_ 

\ 

■^ 

Jj 

/.. 

/ 

FiQ.  20. — Series  16,  Bhowing  movement  in  stomata  of  onion  (A)  and  upper  stomata 
of  alfalfa  (B). 

The  stomatal  movement  in  onion  resembles  that  of  alfalfa  in  a 
number  of  respects.  Both  open  and  close  and  exhibit  day  closure 
and  night  opening  at  much  the  same  time.  On  the  other  hand,  the 
stomata  of  alfalfa  showed  a  much  greater  day  closure  and  a  corre- 
spondingly greater  night  opening.  The  leaves  of  onion  have  a 
larger  amount  of  water  relatively  and  are  less  affected  by  the  factors 
of  evaporation  in  consequence,  with  the  result  that  the  stomata  show 
less  day  closure.  The  speed  of  day  closure  and  secondary  opening  is 
greater  in  the  stomata  of  alfalfa,  as  well  as  the  degree  of  opening. 
The  curve  of  stomatal  movement  in  onion  is  more  irregular  than 
that  in  alfalfa,  as  the  stomata  open  or  close  rapidly  for  a  time,  then 
slowly  or  not  at  all,  and  then  more  rapidly  again.  The  forenoon 
maximum  of  80  per  cent  on  July  27  is  referred  to  as  unstable,  because 
of  a  similar  irregularity.  The  stomata  opened  to  80  per  cent  at 
6  a.  m.,  closed  to  60  per  cent  the  next  hour,  opened  again  to  80  per 
cent  at  8  a.  m.,  closed  to  70  per  cent  at  9  a.  m.  and  opened  once  more 
to  80  per  cent  at  10  a.  m.  Such  behavior  occurred  to  some  extent 
in  all  plants  under  certain  conditions,  but  most  commonly  in  succu- 
lent plants,  such  as  onion  and  Portulaca  oleracea,  and  to  a  lesser 
extent  in  sugar-beet,  cow-beet,  Rumex  patentia,  salsify,  cabbage,  and 
others.  However,  under  certain  conditions,  such  as  high  evapora- 
tion counteracted  by  high  water-content,  alfalfa,  sweet-clover,  corn, 


DAILY   MOVEMENT   IN    CEREALS.  39 

nasturtium,  and  similar  thin-leaved  plants  exhibited  striking  irregu- 
larities in  their  stomatal  movement. 

Under  extreme  conditions  of  temperature,  evaporation,  and  usually- 
low  water-content,  the  stomata  of  the  onion,  as  well  as  those  of 
alfalfa,  were  open  all  night  and  closed  during  the  day.  On  the  other 
hand,  a  very  high  water-content  causes  the  stomata  of  onion  to 
remain  continuously  open  throughout  a  24-hour  period,  opening 
somewhat  wider  upon  sunrise  and  closing  very  slowly  during  the 
night,  but  not  to  less  than  75  per  cent  as  a  rule.  Upon  the  appearance 
of  sufficient  light  at  dawn  such  closure  ceases,  and  the  stomata  again 
open  to  maximum  just  after  sunrise.  However,  even  such  a  plant, 
during  a  very  hot  and  dry  day,  will  show  considerable  midday  closure, 
this  sometimes  being  complete.  Hence,  evaporation  may  cause  the 
stomata  of  onion  to  be  closed  during  the  day  and  open  only  at  night, 
more  or  less  regardless  of  the  water-content,  but  as  this  decreases 
the  effect  of  evaporation  increases,  and  this  reversal  of  normal 
behavior  in  the  stomata  becomes  the  more  usual  occurrence. 

THE  DAILY  MOVEMENT  IN  CEREALS. 

Cereals  are  alike  not  only  in  the  structure  and  mechanism  of  their 
stomata,  but  in  the  behavior  of  these  as  well.  Certain  peculiarities 
which  distinguish  them  from  other  plants  must  be  dependent  upon 
the  unique  structure  of  their  stomata,  but  other  characteristics  found 
in  the  behavior  are  perhaps  due  to  differences  in  the  plant  as  a  whole. 
The  failure  to  show  night  opening  must  be  largely  a  matter  of 
stomatal  mechanism,  but  the  rare  occurrence  of  maximum  opening  of 
all  the  stomata  of  a  cereal  is  probably  the  result  of  peculiarities 
in  other  parts  of  the  plant.  Moreover,  the  rarity  of  maximum 
opening  is  perhaps  a  regional  phenomenon,  as  the  conditions  under 
which  wide  opening  does  occur  show  that  it  is  probably  common  in  a 
more  humid  region,  such  as  the  northeastern  States. 

The  stomatal  movement  described  is  the  average  of  that  found  in 
the  upper  and  lower  surfaces.  The  difference  between  upper  and 
lower  stomata  was  slight  when  it  did  occur  and  was  clearly  due  to 
the  light  intensity.  Hence,  when  a  leaf  was  blown  about  and  alter- 
nately illuminated  on  the  two  sides,  as  usually  was  the  case,  no 
difference  in  the  movement  of  stomata  on  the  two  surfaces  could  be 
detected.  The  two  sets  of  epiderm  were  collected  from  each  plant 
and  the  apertures  were  calculated  from  both  surfaces  to  furnish 
the  graphs.  In  photographing,  however,  only  the  lower  surface  was 
used,  since  this  represented  the  apertures  for  both  surfaces. 

A  set  of  barley  epiderm  as  well  as  of  alfalfa  was  collected  in  series 
1  (fig.  2).  The  stomata  of  barley  were  but  12  per  cent  open  at  9  a.  m., 
when  the  series  started,  and  were  closing,  while  those  of  alfalfa  were 
opening  rapidly.    At  10  a.  m.  barley  showed  less  than  3  per  cent 


40 


THE   DAILY   MARCH    OF   STOMATAL   MOVEMENT. 


opening  and  complete  closure  at  11  a.  m.,  when  the  stomata  of 
alfalfa  reached  maximum.  The  stomata  of  barley  remained  closed 
to  the  end  of  the  series.  The  12  per  cent  opening  at  the  start  of  the 
series  is  the  average  of  200  stomata,  several  of  which  were  at  max- 
imum and  a  great  many  closed.  It  is  not  definitely  known  whether 
a  few  stomata  with  more  accessible  water-supply  do  the  opening  on 
days  of  unfavorable  conditions,  or  whether  groups  of  stomata  open  and 
shut  very  rapidly  and  at  different  times.     Direct  observations  on  the 

same  leaf  would  indicate  the 


30 


20 


\ 

s 

B 

A 

9       10      II     NOON   12        3       4-5 

Fig.  21. — Series  2,  showing  movement  in  lower 
stomata  of  oats  (A)  and  of  barley  (B). 


former,  but  the  fact  that  open 
and  closed  stomata  occur  in 
groups,  and  that  the  stomata  of 
cereals  can  open  and  close  with 
amazing  rapidity,  makes  the 
latter  hypothesis  possible. 

Oats    and    barley   epiderms 
were   collected   in  series  2   as 


well  as  that  of  alfalfa.  The  weather  data  of  this  series  are  shown 
in  figure  3.  Because  of  the  cool,  cloudy  forenoon,  the  barley  stomata 
were  17  per  cent  open  at  9  a.  m.  and  10  per  cent  the  following 

hour.  As  in  the  first  series 
made  the  day  before,  they 
closed  before  11  a.  m.  and 
remained  closed  to  the  end 
of  the  series.  The  stomata 
of  oats  did  not  open  as 
widely  as  those  of  barley, 
but  remained  open  longer. 
They  were  10  per  cent  open 
at  9  a.  m.  and  remained  in 
this  condition  an  hour.  At 
11  a.  m.  they  closed  to  4  per 
cent  and  were  completely 
closed  by  noon.  Like  the 
stomata  of  barley,  they  re- 
mained closed  to  the  end  of 
the  series  (fig.  21).  The  dif- 
ference in  the  weather  con- 
ditions in  the  two  series, 
although  made  on  succes- 
caused  the  stomata  of  barley  as  well  as  those  of  alfalfa  to 


100 
90 
80 

70 
60 
50 
4-0 
30 

zo 

10 


8      9      10      M     NOON    12       3      4-5 

Fig.  22. — Series  3,  .showing  movement  in  lower  stomata 
of  oats  (A)  and  of  barley  (B),  and  upper  stomata  of 
alfalfa  (C);  sunlight  (D),  temperature  (E). 


p 

1  P' 

\, 

'K 

\, 

/ 

■  V 

E 

A 

V 

-/' 

'(^ 

^ 

y 

r~ 

-\ 

7 

\ 

\ 

/ 

/A 

>  \ 

V 

/ 

h 

\ 

/ 

/ 

••• 

B  ^ 

sive  days 

act  differently.  The  same  plants  were  included  in  series  3,  made  3 
days  later,  May  12,  1916.  It  was  a  day  of  passing  clouds,  and  was 
distinctly  cool,  as  the  temperature  did  not  rise  above  63°  F.  Barley 
had  the  same  type  of  stomatal  movement  as  in  the  first  series.    Oats 


DAILY  MOVEMENT   IN   CEREALS. 


41 


showed  16  per  cent  opening  at  9  a.  m.,  6  per  cent  at  10  a.  m.,  and 
showed  closed  stomata  after  this  time,  as  in  barley  (fig.  22).  Although 
conditions  were  different,  the  stomata  of  barley  behaved  in  the  same 
manner  as  in  the  first  series.  However,  it  is  improbable  that  such 
similarity  would  have  been  found  throughout  had  the  two  series  been 
complete.  Other  experiments  have  shown  that  low  morning  temper- 
ature has  an  inhibiting  effect  upon  opening  of  stomata. 

Wheat,  oats,  and  barley  were  included  in  series  10,  June  8  and  9, 
1916,  together  with  alfalfa  and  potato,  the  stomatal  movements  of 
which  have  already  been  described  in  detail.  The  day  was  clear 
and  fairly  warm,  the  highest  temperature  (88  °F.)  being  reached  at 
2  p.  m.  (fig.  7).  Because  of  the  low  humidity,  the  evaporating  power 
of  the  air  was  high,  and  in  consequence  all  the  plants  faced  the  danger 
of  excessive  water-loss.  The  stomata  of  alfalfa  were  closed  for  a 
longer  period  during  the  day  and  hardly  showed  maximum  opening 
at  all.  Potato,  on  the  other  hand,  had  stomata  open  widely  all  day 
and  most  of  the  night.  The  cereals  did  not  have  the  high  water- 
content  in  their  plots  that  the  potato  had,  and  it  is  doubtful  whether 
their  roots  had  been  able  to  follow  the  rapid  dropping  of  the  water- 
table  to  6  or  more  feet.  Hence  the  water-content  of  16  per  cent 
found  at  a  depth  of  2  feet  probably  represented  the  highest  amount 
within  reach  of  the  roots.  Barley  showed  only  a  small  fraction  of 
maximum  stomatal  opening  during  the  first  3  hours  of  daylight  and 
closure  during  the  remainder  of  the  series.  The  greatest  opening 
occurred  at  7  a.  m.,  but  was  only  9  per  cent.  The  stomata  of  wheat 
remained  open  until  after  10  a.  m.,  as  did  the  stomata  of  oats,  but 
while  wheat  reached  22  per  cent  opening  at  7  a.  m.,  that  of  oats  was 


I 

s 

" 

/ 

r 

<, 

^ 

t^ 

t 

>5^ 

=^ 

^it. 

-J 

A       5      6      7      8      9      10      II  NOON   I       234-56789      10     II     MT.     I       234- 
Fig.  23. — Series  10,  showing  movement  in  lower  stomata  of  wheat  (A),  barley  (B),  and  oats  (C). 

only  9  per  cent.  In  none  of  these  plants  was  there  any  indication 
whatever  of  night  opening.  The  results  show  that  wheat  was  appar- 
ently more  able  to  withstand  the  adverse  effect  of  higher  tempera- 
tures and  the  high  evaporating  power  of  the  air  than  either  oats  or 
barley.  Series  7  and  8  show  that  the  stomata  of  barley  do  not  open 
as  widely  or  as  long  when  the  temperature  rises  above  75°  to  80°  F. 
Further  evidence  of  this  is  found  in  series  11,  begun  at  noon  June 
19  and  ended  at  2  p.  m.  June  20,  1916.  Among  the  sets  of  epiderm 
collected  was  one  each  of  wheat,  oats,  barley,  corn,  and  millet.  The 
maximum  temperature  reached  on  June  19  was  79°  F.  and  on  June 
20,  76.5°  F.     The  humidity  was  rather  high  for  the  region,  as  it  at 


42 


THE    DAILY    MARCH    OF    STOAIATAL   MOVEMENT. 


no  time  dropped  below  20  per  cent.  Except  for  the  passing  of  some 
thin  and  hazy  clouds,  the  sunlight  was  nearly  normal,  and  at  no 
time  was  the  light  reduced  to  an  extent  sufficient  to  affect  the  stomata. 
The  weather  data  are  shown  in  figure  16.  At  the  beginning  of  the 
series  the  stomata  of  wheat,  barley,  and  oats  were  closed  and  showed 
no  opening  until  6  a.  m.,  June  20.  Maximum  opening  for  the  morn- 
ing occurred  at  7  a.  m.,  and  was  21  per  cent  in  wheat,  16  per  cent  in 
oats,  and  17  per  cent  in  barley.  Total  closure  occurred  in  all  three 
at  nearly  the  same  time  and  was  complete  at  11  a.  m.  (fig.  24).     In 


, 

A/3^a 

L 

^ 

L 

Fl^; 

_J 

ONI  23-*56  789  10  1IMT.I  234-56789  10  II  NOON  I 
Fig.  24. — Series  11,  showing  movement  in  lower  stomata  of  wheat  (A),  oats  (B),  and  barley  (C). 

comparing  these  results  w^ith  those  obtained  in  series  10,  it  will  be 
observed  that  the  stomata  of  wheat  show^ed  very  little  difference  in 
behavior,  those  of  oats  opened  more  widely  than  on  June  8,  while  those 
of  barley  opened  to  twice  the  width  and  remained  open  2  hours 
longer.  The  chief  difference  in  weather  conditions  during  the  two 
series  was  in  temperature,  the  maximum  on  June  20  being  76.5° F., 
w^hile  the  maximum  of  June  8  was  88".  In  addition,  the  humidity 
was  not  as  low  and  the  evaporating  power  of  the  air  was  correspond- 
ingly less.  Wheat  was  therefore  but  little  affected  by  the  higher 
temperature  of  the  preceding  series,  barley  showed  the  greatest 
effect,  and  oats  occupied  an  intermediate  position. 

The  stomatal  movement  in  millet  {Setaria  italica  germanica)  was 
essentially  the  same  as  in  wheat,  the  only  difference  being  a  slightly 
wider  opening  at  7  a.  m.  in  millet.     The  stomatal  movement  in  corn, 


tlllllllllllllliil 


NOON   12       34       5      6       78      9      10     U    MT.     I        23ASfe78si0n   NOON    1       2 

Fio.  25. — Series  11,  showing  movement  in  lower  stomata  of  corn  (A)  and  millet  (B). 

however,  was  distinctly  different.  The  stomata  were  50  per  cent  open 
at  noon  on  June  1 9  when  the  series  started.  They  closed  gradually  and 
completely  by  3  p.  m.  However,  they  immediately  began  to  open 
and  at  6  p.  m.  reached  15  per  cent,  the  maximum  for  the  afternoon. 
They  then  closed  as  slowly  as  they  had  opened,  completing  the  move- 


DAILY   MOVEMENT   IN    CEREALS.  43 

ment  by  9  p.  m.  As  with  the  other  cereals  examined,  the  stomata 
of  corn  remained  closed  throughout  the  night,  but  opened  directly- 
after  sunrise.  The  maximum  opening  of  42  per  cent  for  the  morn- 
ing occurred  at  9  a.  m.  They  remained  in  this  condition  until  10  a.  m. 
and  then  became  closed  by  noon.  At  1  p.  m.  they  had  again  opened 
and  were  80  per  cent  open  at  2  p.  m.  when  the  series  ended  (fig.  25). 

The  stomatal  movement  in  corn  during  this  series  was  remarkable 
in  several  respects.  One  was  the  much  greater  opening  compared 
with  that  of  the  other  cereals,  and  the  comparatively  little  complete 
closure  during  the  hours  of  daylight.  The  stomata  of  corn  differed 
from  those  of  alfalfa  in  opening  for  a  much  longer  period,  but  never 
to  a  maximum.  Another  important  point  is  the  difference  shown 
on  the  two  successive  days  of  the  series.  As  the  maximum  opening 
for  the  morning  usually  occurred  about  9  a.  m.,  the  opening  of  50 
per  cent  observed  at  noon,  June  19,  indicates  that  the  stomata 
were  more  widely  open  some  hours  previous  to  the  start  of  the  series. 
At  all  events,  the  stomata  showed  considerable  opening  during  the 
forenoon  of  June  19  and  but  slight  opening  on  the  afternoon  of  that 
day.  The  next  day  this  course  was  reversed,  the  stomata  showing 
but  moderate  opening  during  the  forenoon  and  twice  as  great  open- 
ing in  the  early  afternoon.  This  can  not  be  fully  explained  by  the 
physical  factors  recorded,  but  was  probably  in  part  the  result  of 
wind  conditions. 

When  conditions  become  more  unfavorable,  corn  stomata  close 
for  increasing  periods  during  the  day.  Such  midday  closure  is  often 
accompanied  by  rolling  of  the  leaf  and  other  evidences  of  wilting. 
Under  extreme  conditions  the  stomata  open  with  the  first  light  of 
dawn  and  close  shortly  after  sunrise,  to  remain  closed  until  the  next 
morning.  If  conditions  are  less  extreme  they  stay  open  until  late 
morning  and  then  close  until  evening,  when  they  open  for  a  few  hours 
before  darkness  sets  in.  Even  under  these  circumstances,  leaf-rolling 
is  very  noticeable  during  the  early  afternoon,  but  when  the  stomata 
are  more  or  less  open  throughout  the  day,  as  in  series  11,  no  trace  of 
rolling  occurs. 

A  collection  of  epiderm  from  potted  plants  of  wheat  growing  in  the 
greenhouse  at  the  University  of  Minnesota  was  made  during  the 
course  of  series  26,  on  May  5,  1917.  Owing  to  the  remains  of  a 
coat  of  whitewash  and  to  soot  from  the  trains  passing  just  below, 
only  17  per  cent  of  the  light  outside  penetrated  through  the  glass. 
This  light  was  a  diffused  sunlight  and  much  more  than  could  pass 
through  a  canvas  covering,  such  as  used  by  Gray  and  Peirce  in  their 
experiments,  or  through  a  north  window,  such  as  used  by  Darwin. 
The  humidity  was  high,  ranging  from  45  per  cent  to  85  per  cent  and 
averaging  70  per  cent.  The  lowest  temperature  recorded  was  55°  F. 
at  4  a.  m.  and  the  highest  72°  F.  at  2^  ZQT  p.  m.    The  day  being  clear 


44 


THE   DAILY   MARCH   OF   STOMATAL   MOVEMENT. 


and  cloudless,  the  behavior  of  the  stomata  was  practically  normal, 
in  spite  of  the  low  Ught  intensity  in  the  greenhouse,  and  the  fluctua- 
tions in  opening  and  closing  coincide  with  those  of  light  intensity. 
At  the  outset,  the  stomata  were  closed  and  did  not  open  until  after 
sunrise.  At  6  a.  m.  they  were  only  5  per  cent  open,  but  the  next  hour 
they  had  opened  to  50  per  cent  and  at  8  a.  m.  were  wide  open.  They 
remained  at  maximum  until  noon  and  then  started  to  close  slowly. 
At  2  p.  m.  they  were  still  80  per  cent  open,  but  with  the  sudden  drop 
in  light  intensity  at  this  time  they  closed  to  50  per  cent  in  the  next 
hour.  Then,  as  the  hght  decreased  very  little  for  a  time,  the  stomata 
closed  only  to  30  per  cent  in  the  next  2  hours.  At  6  p.  m.  they  closed 
to  10  per  cent,  as  there  was  less  than  1  per  cent  light  in  the  green- 
house, and  completely  by  7  a.  m.    They  remained  closed  all  night. 


100 

— 

r 

'" 

■n 

■n" 

/ 

/ 

^ 

\ 

/ 

V, 

,, 

\ 

/ 

\ 

, 

^ 

\ 

i 

\ 

\ 

^-. 

V 

/ 

.?-- 



..... 

-■•• 

-... 

^ 

^ 



_ 

•■■.-. 

— 

L 

_ 

__ 

_ 

_ 

5      6      7      8      9     10 


2      3     4      5      6      7      8      9      10     II     MT.    I       2      3 


Fig.  26. — Series  26,  showing  movement  in  lower  stomata  of  wheat  growing 
in  the  greenhouse  (A);  light  intensity  outside  (B),  inside  (C). 

The  stomatal  movement  of  wheat  in  this  series  may  be  considered 
somewhat  representative  of  all  cereals  under  the  most  favorable  con- 
ditions. The  small  amount  of  light  causing  opening  is  not  remark- 
able for  plants  grown  in  a  greenhouse,  and  hence  adjusted  to  operate 
under  conditions  of  reduced  light  intensity.  The  plants  in  the  Great 
Salt  Lake  region  did  not  show  this  type  of  movement,  which  is  not 
strange,  as  the  average  daily  minimum  humidity  for  June,  July,  and 
August  1916  was  only  10  per  cent.  The  behavior  of  the  individual 
stomata  was  different  in  this  series.  In  those  made  at  Salt  Lake 
City  only  part  of  the  stomata  opened,  thus  making  the  average  open- 
ing for  all  rather  low.  Then,  as  closure  began,  some  stomata  closed 
slightly,  others  completely,  and  some  not  at  all.  As  time  went  on, 
a  larger  proportion  of  closed  stomata  were  found,  until  finally  all 
were  closed.  In  the  greenhouse  series,  however,  far  the  larger  number 
showed  the  same  degree  of  opening  in  each  strip,  and  less  than  a 
fifth  of  their  number  varied  from  this  average,  and  most  of  these 
only  a  httle.  This  adds  evidence  to  the  belief  that,  in  the  series  made 
during  less  favorable  conditions,  some  remained  closed  throughout 
the  24-hour  period,  while  others  showed  all  the  opening  that  occurred. 


DISCUSSION   OF  RESULTS.  45 

DISCUSSION  OF  RESULTS. 

The  plants  described  are  but  a  few  of  those  investigated,  but  they 
are  representative  in  some  measure  of  all.  Their  stomatal  behavior 
provides  a  basis  for  classifying  the  stomata  of  all  the  species  into  three 
general  groups,  typified  by  barley,  alfalfa,  and  potato.  Naturally, 
the  stomatal  movement  in  each  species  varies  in  some  respects 
from  that  in  the  type,  as  has  been  shown.  Barley  represents  the 
cereal  type  with  stomata  of  peculiar  construction  and  great  sensitive- 
ness, which  show  no  opening  at  night,  no  matter  how  slight  the  day 
opening.  Alfalfa  represents  the  group  which,  under  normally  favor- 
able conditions,  have  open  stomata  during  the  day  and  closed  stomata 
at  night,  but  as  conditions  become  less  favorable  show  increasing 
night  opening  and  extended  mid-day  closure.  Potato  belongs  to  the 
group  of  plants  that  normally  have  open  stomata  at  night  under 
favorable  conditions,  and  close  them  only  as  water-content  decreases 
or  evaporation  becomes  greater.  Naturally,  it  is  possible  by  an 
unusual  grouping  of  factors  to  vary  the  behavior  in  many  plants  to 
such  an  extent  that  they  will  show  almost  any  kind  of  movement. 
For  this  reason  it  is  unsafe  to  carry  out  experiments  in  the  greenhouse 
alone,  as  the  factors  present  differ  greatly  in  degree  from  those  in  the 
field,  and  the  stomatal  movements  of  greenhouse  plants,  in  conse- 
quence, are  distinctly  different.  Hence  greenhouse  experiments  are  of 
value  only  in  connection  with  similar  ones  made  in  the  field. 

Barley  has  never  been  found  in  the  course  of  the  experiment  to  have 
open  stomata  at  night  correlated  with  day  closure.  Many  series 
indicate  that  highly  favorable  conditions  would  probably  cause 
barley  or  corn  to  show  stomatal  behavior  resembling  that  of  wheat 
in  series  26,  i.  e.,  opening  with  the  appearance  of  dayhght,  closing 
gradually  during  the  afternoon,  and  remaining  closed  all  night. 
Under  conditions  only  slightly  less  favorable,  the  stomata  of  barley 
have  been  found  to  close  in  the  afternoon,  and  on  a  hot,  dry  day  were 
but  partially  open  an  hour  or  two  after  sunrise.  In  all  cereals  the 
tendency  seems  to  be  to  operate  with  many  closed  stomata  at  nearly 
all  times.  Even  under  very  favorable  field  conditions  all  the  stomata 
are  wide  open  for  only  an  hour  or  two.  In  the  case  of  corn,  sorghum, 
and  Sudan  grass,  very  warm  or  even  hot  weather,  brilliant  sunshine, 
and  a  high  water-content  seem  to  be  the  optimum  conditions.  For 
wheat,  oats,  and  barl,ey,  cool  and  rather  humid  weather  and  less 
sunshine  are  best.  This  has  a  practical  bearing  on  the  spread  of 
wheat  rust,  as  the  parasite  gains  entrance  to  the  leaves  of  the  host 
through  the  stomatal  openings  and  in  part  explains  how  it  can  make 
most  headway  during  such  weather.  Millet  occupies  a  position 
between  barley  and  corn  in  respect  to  optimum  conditions.  The 
cereal  type  of  stomatal  behavior  may  be  characterized  as  showing 


46 


THE   DAILY   MARCH   OF   STOMATAL   MOVEMENT. 


no  night  opening  correlated  with  mid-day  closure,  and  as  rarely 
showing  maximum  opening  of  all  the  stomata. 

The  alfalfa  t}rpe  of  stomatal  movement  is  characteristic  of  most 
thin-leaved  mesophj-tes.  The  normal  light -curve  of  alfalfa  (fig.  6) 
shows  stomatal  movement  under  the  most  favorable  conditions,  and 
is  the  same  kind  of  curve  as  that  produced  by  a  cereal  under  such 
conditions.  Then,  as  these  become  progressively  less  favorable, 
mid-day  closure  appears,  increasing  to  complete  closure,  which  in 
turn  becomes  more  and  more  prolonged  until  the  stomata  are  closed 
all  day.  With  the  appearance  of  mid-day  closure,  night  opening  also 
develops  and  increases  with  increase  of  day  closure.  The  final  result 
is  a  partial  opening  of  stomata  all  night  and  complete  closure  all  day. 

The  plants  studied  with  this  type  of  movement  are: 


Melilotus  alba,  sweet  clover. 
Trifolium  pratense,  red  clover. 
T.  repens,  white  clover. 
Pisuni  sativum,  garden  pea. 
Lathyrus  odoraius,  sweet  pea. 
Phaseolus  nanus,  navy  bean. 
P.  lunatus,  lima  bean. 
Beta  vulgaris,  sugar-beet. 
B.  vulgaris,  blood-beet. 
Chenopodiwn  album,  lamb's-quarters. 
Rumex  patientia,  western  dock. 
R.  crispu^,  curly  dock. 
Brassica  napus,  rape. 
B.  campestris,  turnip. 
Raphxinus  sativum,  radish. 
Brassica  campestris,  rutabaga. 
Papaver  snmnijerum,  poppy. 
TropcEolum  major,  nasturtium. 


Amaranthus  retroflexu^,  pigweed. 
A.  grcedzans,  little  pigweed. 
A.  blitoides,  carpet-weed. 
Polygonum  aviculare,  knotweed. 
Malta  rotundifolia,  cheeses. 
Pastinaca  sativa,  parsnip. 
Daucus  carota,  carrot. 
Lycopersicum  esculentum,  tomato. 
Citrullus  vulgaris,  watermelon. 
Cucumis  melo,  muskmelon. 
C.  sativum,  cucumber. 
Helianthus  annuus,  sunflower. 
Arctium  lappa,  burdock. 
Lactuca  scariola,  prickly  lettuce. 
L.  sativa,  lettuce. 
Taraxacum  officinale,  dandelion. 
Ribes  aureum,  golden  currant. 
R.  rnhrum,  common  currant. 


The  trees  whose  stomatal  movement  was  investigated  were  apple 
(Malus  sylvestris),  pear  (Pirus  communis),  Elberta  peach  {Prunus 
persica),  sweet  cherry  (Prunus  cerasus),  and  Lombardy  poplar 
(Populus  nigra  italica).  These,  as  well  as  California  privet  (Ligus- 
irumjaponicum),  must  be  classified  with  the  alfalfa  group,  since  night 
opening  does  not  occur  under  favorable  conditions.  On  the  other 
hand,  no  mid-day  closure  was  found  in  any  of  the  series,  as  they 
seemed  to  be  little  affected  by  evaporation.  It  is  impossible  to  state 
the  water-content  about  their  roots,  but  there  is  good  reason  to 
beUeve  that  the  soil  was  moist  at  all  times.  The  behavior  of  their 
stomata  resembled  the  normal  light-induced  movement  in  alfalfa  at 
the  times  when  the  stomata  of  alfalfa  showed  considerable  day  closure. 
This  was  probably  due  in  large  measure  to  the  great  balance  of  water 
on  hand  in  the  trunk  and  branches  of  the  tree,  and  to  the  high 
water-content  within  reach  of  the  roots. 

The  third  group  contains  the  larger  number  of  fleshy-leaved 
plants  as  well  as  some  thin-leaved  ones.    Under  conditions  of  high 


DISCUSSION    OF   RESULTS  47 

water-content  and  low  evaporation,  the  stomata  are  continuously 
and  often  widely  open  all  day  arid  night.  As  the  evaporating  power 
of  the  air  increases  beyond  a  certain  point,  the  stomata  tend  to  close. 
Based  upon  the  time  of  such  closure,  three  subgroups  may  be  dis- 
tinguished. Potato  stomata  at  first  close  just  after  sunset  for  a 
time,  but  as  the  water-content  decreases  this  time  extends  backward 
into  the  afternoon.  Then,  as  conditions  become  extreme,  the  stomata 
close  a  short  time  after  sunrise,  accompanied  by  visible  wilting  of  the 
entire  plant,  which  persists  throughout  the  day  After  sunset  the 
plant  recovers,  the  stomata  open  slowly  about  midnight,  and  at 
sunrise  are  fully  open.  A  second  variation  is  found  in  cow-beet, 
where,  under  favorable  conditions,  the  stomata  are  wide  open  during 
the  day  and  close  very  little  during  the  night.  At  sunrise  this 
closure  is  arrested  and  shortly  afterward  the  stomata  again  become 
wide  open.  As  conditions  grow  less  favorable,  night  closure  becomes 
more  rapid  and  is  completed  before  sunrise.  Progressively  as 
evaporation  increases  and  water-content  decreases,  such  closure  is 
begun  at  an  earlier  time,  becoming  complete  at  midnight  and  then  at 
sunset.  Finally,  the  stomata  open  widely  only  an  hour  after  sunrise 
and  close  gradually  and  completely  during  the  forenoon  or  even  before 
morning  is  over.  The  third  variation  of  this  form  of  movement 
occurs  in  onion,  where  the  stomata  are  wide  open  at  night  under 
conditions  of  high  water-content  and  low  evaporation.  If  water- 
content  alone  becomes  low,  the  stomata  close  at  night,  but  if  the 
evaporation  increases  instead,  the  stomata  tend  to  close  during  the 
day.  Hence,  with  medium  or  low  water-content,  onion  stomata  react 
to  increased  evaporation  like  the  plants  in  the  alfalfa  group,  showing 
increasing  mid-day  closure  correlated  with  increasing  night  opening. 

The  plants  having  stomatal  movement  like  that  of  the  cow-beet 
and  potato  are  cabbage  (Brassica  oleracea),  tulip  (Tulipa  gesneriana, 
Red  Prince),  Portulaca  oleracea,  and  probably  Encelia  farinosa.  This 
latter  plant  has  not  been  found  with  open  stomata  at  night  in  any 
series,  since  the  latter  were  not  made  under  the  necessary  conditions. 
The  general  behavior  is  essentially  like  that  found  in  cow-beet,  and  it  is 
probable  that  under  favorable  conditions  this  plant  would  show  open 
stomata  at  night.  Verbena  ciliata  is  also  included  here,  as  plants 
which  were  heavily  watered  showed  the  stomata  20  per  cent  open  all. 
night.  Fouquiera  splendens  exhibits  a  rather  bewildering  behavior,, 
the  stomata  of  the  primary  leaves  of  heavily  watered  plants  showing 
movement  like  that  of  cow-beet,  while  the  secondary  leaves  showed 
mid-day  closure  and  correlated  night  opening,  such  as  found  in  alfalfa. 

The  plants  with  behavior  similar  to  that  of  onion  are  salsify 
(Tragopogon  porrifolius),  Hubbard  squash  (Cucurbita  maxima), 
crook-neck  squash  (C  moschala),  pumpkin  (C.  pepo),  plantain 
{Plantago  major),  lily  {Lilium  speciosum),  and  leek  {Allium  porrum). 


48  THE   DAILY   MARCH   OF   STOMATAL   MOVEMENT. 

Such  plants  as  Scirpus  validus,  Equisetum  hiemale,  and  E.  palusire 
showed  the  stomata  continuously  wide  open,  and  this  seems  to  be 
their  normal  state.  Equisetum  was  found  with  wide-open  stomata 
during  wilting  and  even  after  the  death  of  the  stems  from  water-loss. 

SUMMARY. 

1.  The  daily  march  of  stomatal  movement  varies  more  or  less 
from  day  to  day.  It  is  as  unusual  to  find  movement  identical  on  two 
successive  days  as  it  is  for  the  weather  to  be  the  same.  This  varia- 
tion is  correlated  with  changes  in  weather  and  water-content,  and 
does  not  occur  when  these  are  sufficiently  alike. 

2.  In  nearly  all  plants,  stomatal  opening  is  correlated  with  the 
presence  of  light  when  conditions  are  favorable.  When  they  become 
unfavorable,  the  influence  of  Ught  is  modified  by  the  action  of  other 
factors,  and  finally  nullified.  In  a  few  plants,  light  seems  to  have 
little  or  no  part  in  producing  stomatal  opening. 

3.  Plants  fall  into  three  groups,  according  to  their  stomatal  be- 
havior, each  of  which  has  several  subdivisions. 

4.  The  first  group  includes  the  cereals  in  which  night  opening 
does  not  occur  under  ordinary  conditions,  favorable  or  unfavorable. 
Day  opening  is  dependent  in  duration  and  degree  upon  favorable 
conditions  of  evaporation,  temperature,  and  water-content. 

5.  The  second  group  includes  most  thin-leaved  mesophytes,  such 
as  alfalfa.  Under  favorable  conditions  their  stomata  are  open  all 
day  and  closed  all  night.  Alfalfa  stomata  open  in  2  to  6  hours  after 
daylight,  remain  open  from  3  to  6  hours,  and  then  gradually  close 
during  a  period  about  twice  as  long  as  required  for  opening.  When 
conditions  become  less  favorable,  the  stomata  close  partially  or 
completely  for  a  time  during  the  middle  of  the  day,  the  period  in- 
creasing to  include  the  whole  day  under  very  unfavorable  conditions. 
Night  opening  appears  when  mid-day  closure  occurs  and  increases  in 
degree  and  extent  with  it.  Finally,  when  conditions  become  extreme, 
the  stomata  are  closed  all  day  and  open  all  night,  the  degree  of 
opening  being  dependent  upon  the  water-content. 

6.  The  third  group  of  plants,  which  includes  the  potato,  tends  to 
have  stomata  open  to  a  greater  or  less  degree  throughout  the  day 
and  night  under  optimum  conditions,  especially  of  water-content. 
If  evaporation  increases  to  a  critical  degree,  the  stomata  close  for  a 
time  during  the  day,  when  this  is  greatest.  If  water-content  de- 
creases moderately,  the  stomata  not  only  become  more  responsive 
to  evaporation,  but  in  most  plants  to  light  changes  as  well.  Hence 
the  stomata  open  with  the  appearance  of  daylight  and  close  at  night, 
unless  evaporation  has  become  serious.  If  the  water-content  de- 
creases to  a  critical  degree,  the  effect  of  sunlight  can  be  modified  or 


SUMMARY.  49 

even  nullified  by  increase  of  evaporation,  thus  producing  day  closure 
and  night  opening,  as  in  the  alfalfa  group  grown  under  similar 
conditions. 

7.  The  stomatal  movement  of  each  plant  studied  tends  to  follow 
a  regular  course  under  optimum  conditions,  opening  and  closing 
progressing  smoothly  and  uniformly  until  complete.  As  conditions 
become  less  favorable,  however,  the  rate  of  movement  becomes  more 
and  more  irregular,  opening,  for  example,  progressing  rapidly  one 
hour,  slowly  or  not  at  all  the  next,  and  rapidly  again  the  third. 
When  evaporation  becomes  extreme,  movement  consists  of  alternate 
opening  and  closing,  the  one  following  the  other  at  hour  or  even 
shorter  intervals.  If  the  general  trend  is  toward  opening,  the  degree 
of  each  opening  exceeds  that  of  closing,  but  if  toward  closing,  the 
reverse  is  true.  The  amplitude  of  these  changes  is  rarely  great  and 
a  difference  of  40  per  cent  from  the  smoothed  curve  is  unusual. 

8.  The  stomata  of  the  upper  and  lower  surfaces  of  the  leaves  in 
most  plants  are  different  in  their  structure  or  in  their  relation  to  the 
rest  of  the  leaf.  Hence  the  stomatal  movement  is  dissimilar,  even 
though  each  surface  be  exposed  to  the  same  environment.  However, 
in  some  plants,  such  as  the  cereals  studied  and  the  sugar-beet,  the 
stomata  are  sufficiently  alike  in  structure  and  in  relation  to  the  other 
tissues  to  produce  like  behavior  under  identical  conditions.  In  some 
of  the  cereals  with  leaves  blown  about  and  alternately  illuminated  on 
each  surface,  the  stomatal  behavior  is  similar  under  most  conditions. 
In  other  cereals  and  in  sugar-beet,  differences  in  illumination  and  in 
exposure  to  other  factors  normally  cause  considerable  divergence  in 
the  behavior  of  the  upper  and  lower  stomata,  although  this  is  not 
as  great  as  in  those  plants  with  dissimilar  stomata. 

9.  The  stomata  on  the  stems,  when  such  are  present,  usually  differ 
materially  from  those  of  the  leaves  in  structure  and  relation  to  water- 
supply,  and  as  a  consequence  in  their  behavior  as  well. 

10.  The  marsh  plants  studied  have  permanently  open  stomata. 


II.  THE  EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT 
CONDITIONS  UPON  STOMATAL  MOVEMENT. 

The  effect  of  stimuli  upon  stomata  has  received  by  far  the  great- 
est attention  from  investigators  in  this  field.  The  problem  is  so 
vast  that  no  attempt  was  made  to  cover  it  in  detail,  but  certain  facts 
have  been  brought  out  in  the  course  of  the  experiments  which  aid 
in  explaining  the  behavior  of  stomata.  Hence,  the  discussion  is 
confined  to  the  previous  series  described  in  their  relation  to  physical 
factors,  and  to  other  series  and  experiments  designed  to  afford  an 
explanation  of  some  of  the  phenomena  observed.  It  is  in  this  con- 
nection that  greenhouse  experiments  are  particularly  valuable  in 
supplementing  field  observations. 

Some  factors,  such  as  light,  have  a  direct  effect  upon  stomatal 
movement,  others,  like  relative  humidity,  have  an  indirect  effect  by 
acting  upon  the  leaf  as  a  whole,  or  like  wind  by  increasing  the  effect 
of  some  other  factor.  The  rate  of  change  in  a  factor  may  have  an 
effect  when  rapid,  and  little  or  none  at  all  when  slow.  Minor  changes 
of  a  factor  have  little  or  no  effect,  except  at  the  critical  point.  Thus, 
changes  in  light  intensity  have  no  observable  direct  effect  if  they  occur 
above  50  per  cent  of  maximum  for  the  region,  but  produce  correspond- 
ing changes  in  stomatal  movement  in  many  plants  growing  where 
the  light  is  less  than  10  per  cent.  There  is  always  a  lag  between  the 
impact  of  a  factor  and  the  resultant  effect  upon  the  stomata.  This 
lag  is  of  variable  length  and  depends  largely  upon  temperature  and 
degree  of  impact,  but  often  upon  other  conditions  as  well,  such  as 
degree  of  maturity,  fatigue,  momentum,  and  structure  and  condition 
of  the  leaf.  As  the  structure  and  condition  of  the  plant  and  its  parts 
also  have  an  effect  upon  stomatal  movement,  these  are  considered 
as  well  as  the  physical  factors. 

LIGHT. 

The  importance  of  light  in  causing  stomata  to  open  has  been  known 
for  many  years,  but  the  manner  in  which  it  operates  is  not  yet 
thoroughly  understood.  Lloyd  (1908)  has  shown  that  in  Verbena 
ciliata,  as  well  as  in  other  plants,  starch  almost  wholly  disappears  i/ 
during  the  early  forenoon,  when  the  stomata  are  at  their  widest,  and 
increases  toward  evening  with  the  closing  of  the  stomata.  Iljin 
(1914)  found,  by  means  of  the  plasmolysis  method,  that  when  the 
stomata  were  open  the  guard-cells  had  a  much  higher  osmotic  pressure 
than  the  surrounding  cells,  and  attributed  this  to  the  conversion 
of  the  starch.  The  appearance  of  light  was  considered  by  him  to 
initiate  enzymatic  change  of  starch  into  sugar.     The  greater  concen- 


LIGHT.  51 

tration  of  sap  resulted  in  endosmosis,  which  increased  the  turgor  of  the 
guard-cells  and  in  consequence  caused  opening.  Then,  after  a  time, 
reconversion  into  starch  was  assumed  to  take  place  and  the  stomata 
closed.  In  many  cases  the  starch  is  manufactured  in  the  guard- 
cells,  but  in  some  it  is  not,  when  they  have  no  chloroplasts.  In 
the  latter  type  of  stomata  the  carbohydrates  are  produced  in  the 
chlorenchyma  and  transported  to  the  guard-cells. 

The  starch-content  of  the  guard-cells  was  studied  in  several  of 
the  series,  in  order  to  determine  its  relationship  to  light  intensity 
and  stomatal  movement.  After  several  trials,  the  following  method 
was  adopted  as  showing  most  clearly  the  relative  amount  of  starch 
present  in  the  plastids:  The  strips  in  which  the  stomatal  openings 
had  been  previously  measured  were  washed  free  of  alcohol  and  left 
in  distilled  water  one-half  hour.  They  were  all  trimmed  to  the 
same  size  and  shape,  and  each  placed  for  exactly  5  minutes  in  1  c.  c. 
of  N/5  solution  of  KI  and  I.  The  strips  were  then  washed  immedi- 
ately in  a  large  amount  of  slightly  alkaline  water  to  stop  the  action 
of  the  iodine,  and  mounted  in  glycerine  jelly.  The  shdes  were 
compared  under  the  microscope  and  arranged  in  a  series  according 
to  the  amount  of  starch  estimated  to  be  present,  taking  into  con- 
sideration the  size  and  color  of  grains.  They  were  then  assigned 
numbers  from  zero  to  100,  based  upon  differences  observable  in  the 
slides,  and  these  numbers  were  used  for  the  starch  index.  The  sys- 
tem was  crude  in  many  respects,  but  was  the  most  satisfactory  one 
available,  and  is  believed  to  give  a  general  estimate  of  the  starch 
present  in  the  guard-cells. 

One  attempt  was  made  in  the  case  of  the  stomata  of  cow-beet  to 
use  a  colorimeter  process.  Fifty  stomata  were  dissected  from  a  strip 
of  epiderm  and  placed  in  a  capillary  tube  4  cm.  long  and  1  mm.  in 
diameter.  A  solution  consisting  of  2  grams  KI  and  1  gram  of  iodine 
in  1,000  c.  c.  of  water  was  used  to  stain  the  grains,  the  action  going 
on  for  2  hours.  For  standards,  a  set  of  11  similar  tubes  were  made 
up,  ranging  from  a  KI+I  solution  without  starch,  a  1/10  dilution, 
a  2/10  dilution,  etc.,  to  10/10  suspension  of  soluble  starch  and  KI  +  I 
solution.  The  two  solution-tubes  nearest  in  color  to  the  guard-cell 
suspension  were  compared  several  times  and  the  difference  in  color 
estimated  in  tenths.  It  was  hoped  by  this  means  to  get  a  truly 
quantitative  measure  of  starch,  since  the  amount  in  the  index  tubes 
was  known.  But  the  method  was  abandoned,  because  it  was  very 
difficult  to  get  the  guard-cell  starch  into  suspension,  and  because  the 
differences  between  these  suspensions  were  mostly  too  sUght  to  be 
detected. 

The  starch  index  determined  in  this  manner  did  not  show  the  close 
correlation  to  either  light  changes  or  stomatal  movement  that  Iljin 
and  Lloyd  have  found.     Lloyd  observed  a  clear  and  definite  relation 


52 


EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 


between  changes  in  the  starch-content  of  the  guard-cells  and  sto- 
matal  movement,  but,  as  his  graphs  show,  no  midday  closure  oc- 
curred and  his  curves  are  of  normal  light-induced  movement  only. 
This  type  of  movement  has  also  been  found  correlated  with  changes 
in  the  starch  index,  as  the  following  series  shows :  Series  6  was  begun 
at  8  a.  m.  June  1,  1916,  and  ended  at  9  p.  m.  the  same  day.  At  the 
start  the  stomata  were  but  40  per  cent  open  and  did  not  reach  maxi- 
mum until  noon.  This  slow  morning  opening  was  due  to  the  heavy 
clouds  and  a  drizzling  rain  which  lasted  until  11^  30°"  a.  m.  The 
stomata  remained  at  maximum  until  3  p.  m.  At  4  p.  m.  they  closed 
to  75  per  cent  and  at  5  p.  m.  to  50  per  cent.  No  strips  were  then 
collected  until  8  p.  m.,  when  the  stomata  were  found  closed;  they 
remained  closed  to  the  end  of  the  series.  At  the  outset  the  starch 
index  dropped  rapidly  to  the  minimum  between  11  a.  m.  and  noon 
and  then  rose  slowly  until  3  p.  m.  It  then  rose  rapidly  as  the  sto- 
mata closed  and  continued  rising  to  the  end  of  the  series.  It  still 
showed  a  rapid  increase  at  8  and  9  p.  m.,  although  the  stomata  were 
closed  (fig.  27).     The  stomatal  movement  in  Lombardy  poplar  was 


7 

\ 

/ 

/ 

— 

/ 

-- 

\ 

\ 

/ 

y 

/ 

1 

f 

\D 

■\ 

/ 

/ 

/ 

\ 

/ 

.-: 

\ 

/ 

/ 

\ 

^••V 

..^^ 

"^^ 

t'" 

-\~ 

V 

/ 

/~ 

\7 

— 

"'; 

^v_ 

/. 

/ 

\ 

•t 

"^^ 

\ 

r 

\, 

\ 

^ 

\\ 

...••■' 

\ 

100 
90 
80 
70 
60 
50 
AC 
30 
20 


7 

•••■■ 

i 

c- 

4 

\ 

V 

/ 

\ 

J 

..- 

— 

15' 

■ — 

\ 

1 

A 

' 

\ 

/ 

\ 

y 

\, 

?^ 

1 

\ 

^ 

/ 

9      10 


NOON 


3     4       5      6      7      8      9 


e      9      10     II  NOON  I 


FiQ.  27. — Series  6,  showing  movement  in  lower  stomata  Fig.  28. — Series  7,  showing  movement 

of  Lombardy  poplar  (A),  and  starch  index  in  lower  stomata  of  i2.  pattentio  (A), 

of  the  guard-cells  (B),  sunlight  (C),  humid-  and  starch  index  of  the  guard-cella 

ity  (D),  temperature  (E).  (B),  sunlight  (C),  temperature  (D). 

light-induced,  not  modified  by  any  other  factor,  and  was  correlated 
with  changes  of  the  starch  index  as  Lloyd  found  in  Yerbena. 

Iljin  found  that  when  stomatal  closure  was  induced  by  wilting, 
the  osmotic  pressure  of  the  guard-cells  dropped  and  the  amount  of 
starch  in  them  increased.  No  such  definite  agreement  could  be 
found,  however,  between  midday  closure  and  changes  in  starch- 
content.  In  some  series  the  amount  of  starch  increased  sUghtly 
during  midday  closure,  and  this  was  quite  noticeable  when  closure 
was  protracted.  In  others,  no  corresponding  increase  whatever  was 
found.     Series  7,  Rumex  patientia,  illustrates  this.     The  upper  sto- 


LIGHT.  53 

mata  were  70  per  cent  open  at  8  a.  m.,  90  per  cent  at  9  a.  m.,  and  at 
maximum  the  following  hour.  At  noon  they  were  still  wide  open, 
but  closed  to  20  per  cent  at  2  p.  m.,  remaining  at  this  opening  1  hour. 
Then  they  opened  to  90  per  cent  at  5  p.  m.,  when  the  series  closed 
(fig.  28).  The  starch  index  resembles  that  of  poplar  series  6,  no 
change  having  occurred  because  of  mid-day  closure.  The  amount 
of  starch  in  the  guard-cells  was  decreasing  rapidly  at  the  start  of 
the  series  and  reached  a  minimum  at  10  a.  m.  No  change  was 
observable  the  next  hour,  but  after  this  the  amount  increased  uni- 
formly and  gradually  to  the  end  of  the  series.  Starch  was  on  the 
increase  while  the  stomata  were  closing,  but  hardly  fast  enough  to 
produce  as  great  closure  as  found.  It  was  still  increasing  at  the 
same  rate  when  the  stomata  reopened,  contrary  to  expectations.  It 
is  evident  that  reduced  concentration  of  the  surrounding  cells,  or 
increased  density  in  the  guard-cells  due  to  some  other  factor,  caused 
this  behavior. 

In  some  series  there  was  at  times  a  certain  relationship  between 
changes  in  the  starch  index  and  mid-day  closure,  while  at  other  times 
no  relation  existed.  Thus,  in  alfalfa  during  series  10,  the  stomata 
were  opening  rapidly  at  the  start  and  the  amount  of  starch  was 
decreasing  almost  as  rapidly.  The  observable  minimum  in  the  starch 
index  was  reached  at  7  a.  m.  and  persisted  until  10  a.  m.  In  the 
meantime,  the  stomata  opened  to  the  maximum  at  6  a.  m.,  closed 
to  40  per  cent  at  8  a.  m.,  and  reopened  to  60  per  cent  at  9  a.  m. 
changes  in  starch-content  showing  no  relation  to  this.  After  10  a.  m. 
the  stomata  closed  gradually,  and  completely  at  2  p.  m.  This  is 
correlated  with  a  similar  slow  rise  in  the  starch-content  of  the  guard- 
cells.  They  opened  to  25  per  cent  at  3  p.  m.,  accompanied  by  a 
slight  drop  in  the  amount  of  starch,  and  then  closed  to  10  per  cent 
at  6  p.  m.,  without  change  of  starch-content.  They  remained  sta- 
tionary for  2  hours,  while  the  starch  index  increased  perceptibly.  It 
was  still  increasing,  though  more  slowly,  when  night  opening  started, 
but  between  11  a.  m.  and  1  p.  m.,  when  the  stomata  showed  the 
greatest  increase,  it  remained  stationary.  Between  1  and  3  a.  m., 
when  they  closed  again,  the  starch  index  increased  rapidly  once  more, 
and  decreased  very  much  the  next  hour,  when  morning  opening 
started  (fig.  29).  Hence,  at  times  there  existed  a  good  correlation 
between  the  changes  in  starch-content  and  stomatal  movement  and 
at  other  times  none  whatever.  It  seems  clear  that  the  entire  subject 
is  more  complex  than  either  Iljin  or  Lloyd  has  supposed,  and  must 
be  the  subject  of  renewed  investigation. 

The  time  required  to  produce  response  to  light  of  various  inten- 
sities was  first  studied  at  Salt  Lake  City  in  June  and  July  1916. 
In  order  to  determine  the  reduction  necessary  to  induce  closure  of 
the  stomata,  4  control  cabinets,  used  in  the  sulphur-dioxide  experi- 


54 


EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 


ments,  were  set  up  on  as  many  plots.  These  cabinets  were  6  feet 
square,  4  feet  high,  and  consisted  of  a  light  wooden  framework 
covered  with  sheets  of  celluloid.  A  constant  current  of  air  was 
driven  through  a  6-inch  sheet-iron  pipe  from  an  electric  blower  to  the 
top  of  the  cabinet,  and  given  a  whirling  motion  by  stationary  radial 
vanes  at  the  vent  of  the  pipe  (plate  10).  The  air  escaped  from  the 
cabinet  through  an  adjustable  slot  3  inches  wide  around  the  bottom. 
The  first  cabinet  was  used  unchanged,  the  second  shaded  by  a  large 
muslin-covered  screen,  the  third  covered  with  muslin  tacked  on  above 
the  celluloid,  and  the  fourth  with  oil  fabric  or  imitation  leather,  the 
black  side  turned  in.  Of  the  light  incident,  60  per  cent  entered  the  first 
cabinet,  8.5  per  cent  the  shaded  cabinet,  and  only  2  per  cent  entered 
the  muslin-covered  one.  The  fourth  cabinet  showed  only  the  faintest 
traces  of  light. 


-r       -                    -         H 

iS                         :^-        1 

?  \   /  ^                /           ^ 

l:\z^ik^  /s  a  /-_ 

P           ^^ 

'^X          -.2     4  S^ 

^.       z^y  ^             .^ 

3     4-56 


Fig.  29. — Series  10,  showing  movement  of  upper  stomata  of  alfalfa  (A)  and 
starch  index  of  guard-cells  (B). 

The  cabinets  were  placed  on  alfalfa  plots  to  determine  the  readiness 
with  which  the  stomata  would  respond  to  reduction  of  light  during 
the  forenoon  when  they  had  just  opened,  and  again  in  the  afternoon 
when  they  were  closing.  On  July  3,  1916,  the  cabinets  were  placed 
upon  well-watered  plots  for  90  minutes,  at  3  p.  m.,  when  the  stomata 
were  80  per  cent  open.  After  half  an  hour  the  opening  was  75  per 
cent  in  all  except  the  dark  cabinet.  At  the  end  of  an  hour  it  was  60 
per  cent  in  the  open  and  in  the  unshaded  and  shaded  cabinets,  40 
per  cent  in  the  muslin-covered  cabinet,  and  15  per  cent  in  the  dark 
cabinet.  At  the  end  of  the  experiments  the  opening  was  50  per  cent 
outside  as  well  as  in  the  first  cabinet,  45  per  cent  in  the  shaded 
cabinet,  20  per  cent  in  the  muslin-covered  one,  and  closed  in  the 
dark  cabinet.  The  reduction  of  the  light  to  60  per  cent  had  no  effect 
and  to  8.5  per  cent  but  little.  Reduction  to  2  per  cent  caused  notice- 
able increase  in  the  rate  of  closure,  while  darkness  caused  the  stomata 
to  close  in  less  than  an  hour  and  a  half.  Long-period  experiments  of 
6  to  24  hours  made  at  various  other  times  showed  that  the  reduction 
of  light  in  the  unshaded  cabinet  was  not  sufficient  to  cause  changes. 


LIGHT.  55 

The  difference  in  the  degree  of  reduction  produced  a  difference  in 
the  time  necessary  to  show  an  observable  response,  as  well  as  in  the 
degree  of  response.  In  the  first  half  hour  only  the  plants  inside  the 
dark  cabinet  showed  a  response;  at  the  end  of  an  hour,  the  plants 
in  the  muslin-covered  cabinet  sho^wed  a  response  as  well;  at  the  end 
of  an  hour  and  a  half  the  plants  of  all  the  cabinets  except  the  first 
showed  a  response.  Hence,  with  light,  at  any  rate,  the  difference 
in  time  between  the  impact  of  a  factor  and  the  response  decreases 
as  the  degree  of  change  increases. 

Three  days  later  the  cabinets  were  placed  on  the  plots  at  9^  30"' 
a.  m.  and  removed  at  11  a.  m.  The  stomata  of  the  plants  outside 
were  wide  open  throughout  the  period.  No  change  of  behavior  was 
observed,  except  in  the  dark  cabinet,  in  which  some  closure  had  be- 
gun as  the  experiment  ended.  It  seems  evident  that  the  time  of 
day  also  makes  a  difference  in  the  response,  and  that  stomatal 
movement  may  be  hastened  more  easily  than  reversed.  This  was 
shown  with  even  greater  clearness  in  the  experiments  with  night 
illumination  and  in  those  carried  on  in  April  1917  at  the  greenhouse  of 
the  University  of  Minnesota.  Potted  plants  of  wheat  and  corn  kept 
38  hours  in  darkness  showed  stomatal  opening  in  40  minutes  when 
exposed  to  the  Hght  of  a  40-watt  mazda  light  hung  2  dm.  above  the 
plants,  while  others  kept  in  darkness  3  hours  showed  no  opening 
after  2  hours  of  such  illumination. 

Potted  plants  of  wheat  which  had  been  kept  outside  were  taken 
inside  on  the  evening  of  May  4,  1917,  and  placed  near  those  that  had 
been  grown  in  the  greenhouse  for  series  26.  At  the  start  of  the  series, 
4  a.  m.  May  5,  all  the  plants  were  stripped  at  hour  intervals  until 
9  a.  m.  as  were  the  plants  of  the  series.  The  plants  brought  into  the 
greenhouse  showed  5  per  cent  opening  at  6  a.  m.,  as  did  the  plants 
which  had  been  grown  inside.  At  7  a.  m.  the  greenhouse  plants  had 
stomata  half  open,  while  those  brought  in  were  but  10  per  cent. 
The  next  hour  the  greenhouse  plants  showed  stomata  at  maximum, 
and  the  out-door  plants  35  per  cent  opening.  At  9  a.  m.  both  groups 
of  plants  had  wide-open  stomata.  It  is  evident  that  the  stomata  of 
wheat  plants  accustomed  to  strong  light  do  not  open  as  readily  in 
diffuse  light  as  those  of  plants  adjusted  to  such  light  conditions. 

To  find  the  effect  of  night  illumination  upon  alfalfa  plants  growing 
in  the  field,  a  series  was  made  on  August  13,  1919,  from  midnight 
to  2  a.  m.  A  250-watt  mazda  light  was  suspended  1  meter  above  a 
plot  of  alfalfa  and  a  similar  one  above  a  plot  of  cow-beet.  The  night 
was  clear  and  moonlight,  the  average  temperature  during  the  experi- 
ment being  60°  F.  No  difference  in  stomatal  movement  could  be 
detected  between  the  hghted  and  unlighted  plots,  the  stomata  of 
alfalfa  being  40  per  cent  open  throughout  the  experiment  and  those 
of  cow-beet  remaining  75  per  cent  open  during  the  2  hours.    Hence, 


56         EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 

it  seems  that  the  additional  light  was  not  sufficient  to  cause  further 
opening  in  either  plant,  and  that  the  amount  of  opening  found  was 
caused  by  moonlight  alone. 

Another  series  was  made  August  23,  1919,  to  find  the  effect  of 
such  illumination  on  a  dark  night.  A  plot  of  alfalfa  and  one  of  cow- 
beet  were  illuminated  from  10  p.  m.  to  midnight  and  strips  collected 
every  30  minutes.  The  stomata  of  alfalfa  were  closed  at  the  start 
and  remained  closed  until  11*"  30""  p.  m.  in  the  dark  plot.  In  the 
illuminated  plot  the  upper  stomata  showed  slight  opening  at  11  p.  m. 
and  by  12  had  opened  to  25  per  cent.  Night  opening  also  occurred 
in  the  dark  plot,  but  was  only  5  per  cent  at  midnight.  No  opening 
occurred  in  the  lower  stomata  of  either  plot.  In  the  cow-beet,  the 
stomata  were  40  per  cent  open  at  the  start  of  the  experiment  and 
closed  in  the  unilluminated  plants  to  25  per  cent  at  midnight.  They 
closed  slightly  in  the  upper  surface  of  the  plant  in  the  lighted  plot 
as  well,  since  they  were  35  per  cent  open  at  midnight.  The  lower 
stomata  of  the  lighted  plants  were  like  those  of  the  unlighted  ones. 
Three  hours  later,  from  3  to  4^  30°'  a.  m.  August  24,  the  experiment 
was  repeated  on  plots  that  had  not  been  used  previously.  The 
upper  stomata  of  alfalfa  were  closed  and  remained  closed  in  the  dark 
plot,  while  they  opened  30  per  cent  in  90  minutes  in  the  light  plants, 
the  first  slight  appearance  of  opening  being  produced  in  half  an  hour. 
The  upper  stomata  of  cow-beet  were  20  per  cent  open  at  the  outset 
and  closed  to  15  per  cent  in  the  unlighted  plants,  but  opened  to  50 
per  cent  in  90  minutes  in  the  plants  illuminated. 

These  results  indicate  that  night  illumination  is  effective  on  a  dark 
night,  but  not  on  a  night  which  is  brightly  lighted  by  the  moon. 
The  additional  light  was  not  sufficient  in  the  earlier  experiment  to 
produce  increased  opening.  The  time  of  night  likewise  plays  an 
important  part  in  the  effectiveness  of  illumination,  since  in  alfalfa 
only  25  per  cent  opening  occurred  in  2  hours  during  the  earlier  part 
of  the  night,  while  30  per  cent  was  produced  in  an  hour  and  a  half 
toward  morning.  In  cow-beet  no  opening  occurred  in  the  first  part 
of  the  night,  but  in  the  later  part  rapid  opening  was  found.  This 
may  be  due  either  to  fatigue  or  more  probably  to  the  fact  that  storing 
of  starch  had  to  be  reversed  during  the  first  part  of  the  night,  but 
was  practically  complete  and  everything  ready  for  the  reverse  process 
during  the  later  part  of  the  night. 

TEMPERATURE. 

Temperature  is  manifest  to  a  plant  in  three  forms,  radiant  energy, 
air-temperature,  and  soil-temperature.  As  radiant  energy  it  tends  to 
raise  the  temperature  of  the  leaf  above  that  of  the  surrounding  air, 
and  this  must  be  met  by  the  cooling  action  of  transpiration.  As  air- 
temperature,  it  affects  the  evaporating  power  of  the  air,  and  deter- 


TEMPERATURE. 


57 


mines  in  large  measure  the  speed  of  the  reactions  occurring  in  the 
leaf.  As  soil-temperature,  it  affects  the  direct  water-loss  from  the 
soil  and  the  growth  and  functioning  of  the  roots.  In  all  these  cases 
temperature  plays  an  important  though  indirect  part  in  the  behavior 
of  the  stomata. 

In  one  respect,  however,  temperature  acts  directly  upon  stomatal 
movement,  affecting  the  rate  of  morning  opening.  Such  opening  is 
light-induced  and  hence  dependent  upon  starch  conversion,  which  is 
undoubtedly  an  enzymatic  process.  It  is  to  be  expected  that  such 
conversion  will  therefore  follow  the  same  law  in  regard  to  rate  of 
reaction  as  any  other  chemical  reaction.  No  rigorous  study  of  this 
could  be  carried  out,  but  inspection  of  the  various  series  led  to  the 
conclusion  that  possibly  humidity  changes  and  fluctuations  of  light 
intensity  within  certain  limits  would  not  seriously  affect  the  results 
of  the  experiments  attempted.  These  consisted  in  taking  pots  of 
alfalfa  kept  in  the  dark-room  over  night  into  the  sunlight  on  days 
of  different  temperatures.  It  was  realized  that  it  was  impossible 
to  allow  for  the  effects  of  changes  in  hght,  humidity,  wind,  and  other 
factors,  and  hence  the  experiments  were  liable  to  serious  error,  but 
on  the  whole  the  experiments  agreed  very  well,  and  are  at  least 
indicative  of  the  general  effect  of  temperature  upon  opening  (fig.  30). 

The  plants  were  well-watered 
the  evening  before  and  carried 
into  the  dark-room.  If  condi- 
tions were  of  the  kind  desired, 
they  were  carried  out  and  placed 
in  the  sun  the  next  day  about  9 
or  10  a.  m.  after  stripping  a  leaf 
for  evidence  that  the  stomata 
were  closed.  Strips  were  then 
removed  at  hour  and  half-hour 
intervals  on  cold  days  and  at 
5-minute  intervals  on  very  hot 
days,  in  order  to  determine  the 
time  of  maximum  opening.  There 
was  rarely  more  than  2°F.  dif- 
ference between  the  temperature 
of  the  dark-room  and  the  air  out- 
side, and  hence  no  error  could  be 
expected  from  this  source.     As 

the  light  in  each  experiment  was  at  least  80  per  cent  of  the  max- 
imum for  the  region,  light  differences  could  hardly  introduce  any 
serious  error.  Apparently,  evaporation  had  little  or  no  effect  upon 
this  opening  when  it  was  not  excessive,  causing  closure  before  or 
shortly  after  maximum  opening  occurred.     In  all,  23   experiments 


HOURS 
8.00 


1 

J^ 

r 

A        J 

\ 

F 

30      4-0 

50 

60        70 

80       90 

"C 

6 

vO 

20 

30 

Fig.  30.- 


-Relation  of  speed  of  total  opening 
to  temperature. 


58 


EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 


were  made,  6  of  which  were  discarded  because  too  great  a  change  of 
temperature  occurred  while  the  stomata  were  opening  or  because 
the  stomata  were  not  closed  at  the  start. 

Just  above  freezing,  it  required  8  hours  to  produce  opening.  At 
10°  C.  approximately  4  hours  were  required  to  reach  maximum.  At 
20°  C.  shghtly  less  than  2  hours  was  required,  and  at  30°  C.  nearly  an 
hour.  The  highest  temperature,  34°  C.,  caused  complete  opening 
in  less  than  50  minutes.  At  freezing  and  above  40°  C.  opening  does 
not  occur  or  is  very  erratic.  Hence,  upper  and  lower  limits  for 
stomatal  movement  must  be  near  these  temperatures.  The  results 
obtained  correspond  with  the  time  required  for  opening  by  the 
plants  of  the  series,  these  plants,  however,  requiring  a  somewhat 
longer  time,  since  the  light  intensity  at  the  start  is  low.  This  also 
varies,  as  apparently  at  higher  temperatures  it  requires  less  light 
to  initiate  opening.  If  this  be  true,  it  explains  why  plants  show 
opening  at  the  first  traces  of  approaching  dawn  when  the  weather 
is  hot,  and  no  opening  until  after  sunrise  on  cold  mornings. 

The  effect  of  changes  in  soil-temperature  on  stomatal  movement 
is  difficult  of  determination  for  several  reasons.  During  the  course 
of  a  24-hour  period,  the  superficial  layer  of  dust  in  a  cultivated  field 
undergoes  greater  changes  of  temperature  than  the  air  if  the  day  is 
clear,  but  at  a  depth  of  1  dm.  these  fluctuations  are  not  nearly  so 
great  and  consist  of  a  steady,  slow  rise  after  sunrise  and  a  gradual 
decrease  from  evening  through  the  night.  The  amplitude  of  this 
variation  increases  as  the  soil  becomes  drier,  since  the  conductivity 
of  dry  soil  is  greater  than  that  of  moist.    At  greater  depths  this  slow 


no 

\-^ 

N 

/ 

^ 

N 

^ 

" 

"^ 

v. 

\, 

70 

CO 

^ 

r 

.-- 

._. 

_V 

v: 

—  . 

^ 

'^ 

~- 



— 

— 

— 

-C 

._, 

y 

'./- 

L_ 

-- 

-' 

-• 

\ 

L 

N 

=55 

=>^ 

=5! 

^ 

A 

^ 

/ 

"b" 

— 

.0 

FiQ.  31. — Series  34,  factor  data  for  September  10-11,  1919;  temperature  2  dm. 
above  soil  surface  (A),  at  the  surface  (B),  at  1  dm.  in  the  soil  (C). 

rise  and  fall  becomes  less,  until  at  5  dm.  it  is  very  slight.  At  such  a 
depth  only  a  series  of  cold  days  following  hot  weather,  or  the  reverse 
of  this,  causes  any  distinct  changes  in  temperature.  Naturally,  there 
is  a  gradual  rise  at  this  and  greater  depths  during  the  spring,  and  as 
slow  a  decrease  toward  winter,  but  these  seasonal  changes  do  not 
come  within  the  scope  of  this  discussion.    The  changes  observed  in 


EVAPORATION    AND    CAUSAL    FACTORS.  59 

the  temperature  of  the  air,  the  upper  centimeter  of  soil,  and  at  a 
depth  of  1  dm.  during  the  course  of  series  34  are  shown  in  figure  31. 

Since  the  temperature  of  the  soil  differs  so  much  at  various  depths, 
the  effect  upon  shallow-rooted  plants  can  not  fully  be  determined, 
but  must  play  some  part  in  the  functioning  of  the  roots.  The  tem- 
perature of  the  superficial  layers  also  has  an  effect  upon  the  carpet- 
weeds,  like  Portulaca  oleracea  and  Amaranthus  blitoides,  as  these 
plants  are  subject  to  higher  working  temperatures  than  the  more  erect 
plants.  Deep-rooted  plants,  such  as  alfalfa,  have  their  roots  in  soil 
of  uniform  temperature  and  hence  only  seasonal  changes  affect  them. 

To  determine  the  reaction  of  the  stomata  to  changes  in  soil- 
temperature,  a  number  of  alfalfa  plants  were  exposed  to  sunlight  in 
metal  containers,  containing  the  same  soil  with  the  same  water- 
content.  Some  of  these  were  blackened  to  absorb  more  heat,  some 
left  bright,  and  others  were  placed  in  moist  earth  for  protection.  A 
chemical  thermometer  was  placed  in  the  soil  of  each  container.  The 
results  were  indefinite,  as  sudden  rises  in  temperature  caused  an 
effect  at  lower  temperatures  than  slow  rises  did.  Low  water-content 
increased  the  effect  or  caused  closure  at  lower  temperatures,  and 
other  factors  which  could  only  be  guessed  at  entered  into  the  experi- 
ments. Closure  was  caused  by  temperatures  ranging  from  21°  to 
30°  C.  at  different  times.  It  was  found,  however,  that  a  rise  of  tem-  ) 
perature  in  the  soil  would  first  cause  stomatal  closure  and  then  wilt- 
ing, even  when  the  humidity  was  very  high. 

The  temperature  of  the  leaves  of  a  plant,  while  rarely  the  same, 
usually  differs  but  slightly  from  that  of  the  air.  This  difference  is  the 
result  of  equilibrium  between  the  cooling  effect  of  transpiration  and 
that  of  radiant  energy.  A  number  of  readings  of  leaf-temperature 
were  made  upon  alfalfa,  potato,  and  sugar-beet  with  a  chemical  ther- 
mometer graduated  to  tenths  of  1°C.  The  temperature  of  the  air 
was  read,  then  that  of  a  leaf  wrapped  about  the  bulb,  and  the  air- 
temperature  again.  An  unwilted  alfalfa  leaf  did  not  vary  more  than 
0.2°  C.  above  air-temperature,  but  a  wilted  leaf  was  at  one  time  found 
to  be  1.9°  C.  above  the  temperature  of  the  air.  Usually  the  leaves 
with  open  stomata  were  found  to  be  lower  than  the  air,  and  those 
with  closed  stomata  during  the  day  to  be  higher.  Compared  with 
the  results  obtained  by  E.  B.  Shreve  (1919a)  with  far  more  accurate 
methods,  these  readings  are  too  low,  but  are  indicative  of  the  effect 
of  stomatal  movement  upon  leaf-temperature. 

EVAPORATION  AND  CAUSAL  FACTORS. 

Evaporation  is  a  product  of  several  factors,  the  most  important 
of  which  are  absolute  humidity,  air-temperature,  barometric  pres- 
sure, wind,  and  sunlight.  It  is  usually  measured  as  the  water-loss 
from  a  free  water-surface  or  from  some  type  of  evaporimeter,  the 
most  important  of  which  is  the  Livingston  porous-cup  atmometer. 


60         EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 

Unfortunately,  none  of  these  agree  very  closely  with  the  water-loss 
from  a  plant  or  even  with  each  other,  as  Briggs  and  Shantz  (1917) 
have  shown.  This  is  due  to  the  fact  that  they  are  not  affected  in 
the  same  manner  or  to  the  same  extent  by  each  of  the  factors  con- 
cerned. In  consequence,  attempts  to  calibrate  these  with  one  another 
have  not  met  with  much  success.  Even  in  the  case  of  free  water- 
surfaces  it  has  not  been  possible  to  correlate  the  evaporation  of  one 
with  that  of  another,  except  within  certain  limits  (Thomas  and  Fer- 
guson, 1917).  Since  the  porous-cup  atmometer  is  standardized  and 
in  widespread  use,  it  is  useful  as  a  measure  of  relative  conditions  in 
various  habitats,  but  can  not  be  used  in  studies  of  hourly  water- 
loss  from  the  plant. 

Until  an  evaporimeter  is  devised  the  water-loss  of  which  will 
correlate  accurately  with  the  transpiration  of  a  plant  having  open 
stomata,  it  is  advisable  to  measure  each  factor  by  itself.  It  will  then 
be  possible  to  determine  the  effect  of  changes  in  one  factor  upon  the 
plant  under  various  constant  combinations  of  other  factors.  It  is 
also  possible  to  arrive  at  some  conclusion  concerning  the  effect  of 
any  one  factor  when  all  are  measured  at  hourly  intervals  during  a 
24-hour  period,  or  oftener  when  necessary,  and  compared  with  the 
plant  responses.  This,  in  short,  was  the  manner  of  carrying  out  the 
later  and  more  elaborate  series.  The  factors  concerned  in  evapora- 
tion measured  were  the  temperature  of  the  air,  soil-surface,  and 
soil  at  a  depth  of  1  dm.,  the  wind  velocity,  wet-bulb  depression, 
barometric  pressure,  evaporation  from  a  white-cylinder  porous-cup 
atmometer,  from  a  special  blotting-paper  evaporimeter  standardized 
in  darkness  or  dim  light  against  a  free  water-surface,  and  from  a  free 
water-surface  in  a  flat  pan  blackened  inside.  The  atmometers  were 
standardized  by  the  maker  and  given  the  factors  0.68  and  0.73. 
The  readings  from  these  were  reduced  to  unity. 

The  water-loss  from  the  free  water-surface  was  determined  by 
weighing.  The  pan  was  a  large  Petri  dish  painted  black  on  the  inside, 
and  was  filled  to  a  depth  of  6  mm.,  which  brought  the  surface  to  9mm. 
of  the  edge.  This  level  was  not  permitted  to  drop  below  2  mm.  from 
its  original  height,  when  it  was  replenished  from  an  inverted  flask 
with  burette  tips  and  stopcock,  which  permitted  water  to  be  added 
drop  by  drop  to  the  pan  until  the  exact  original  weight  was  reached. 
The  atmometers  were  not  weighed,  but  were  calibrated  for  changes 
of  temperature,  a  thermometer  running  through  the  cork  into  the 
reservoir  bottle.  The  water-level  of  each  was  brought  to  the  original 
level  marked  on  the  open  tube  once  an  hour,  the  water  being  added 
from  burettes.  This  method  was  checked  carefully  by  weighing  and 
found  accurate  to  0.02  c.  c.  The  blotting-paper  first  used  was  green 
in  color,  matching  the  shade  of  alfalfa  leaves  as  closely  as  possible. 
Later  it  was  found  that  a  standard  blue-green  paper  had  practically 
the  same  rate  of  absorption  of  radiant  energy,  and  this  was  used. 


EVAPORATION   AND    CAUSAL   FACTORS. 


61 


1 

[=--==1  -ft 

\                              /^  -i  -U 

^-^ — --     ^     /-^  n:  T_r  A 

^-^"t  t'^i^ 

^  V"^        ^x        flJ^^    vt^ 

^-7^      -\  crA        fc-  -"-....    I   M 

^-    ..•..x,'f^n            ■■•..      .  n 

t,       .■    ■•■■?      ••■    '■■■.J~^             ■•■■.   ;    ■■■ 

N.::. ■■                          J/   ..-■ 

-It      A      ^ 

tA\A^^ 

y^jyy- 

o        .--i   ■■■■           L^ 

••■■■•••       7              E^ 

^ ■              I           /v 

-       H   ^     fc'"'-:^  ^  - 

:"--^-==-±-¥v^^ 

^--""'               4 

-^-t    ^               s 

'    S^    ^^^^                 _,! 

^!,^    ^:!^          jt 

^SKfSr^V         ]J 

-S  s 

\ 

n 

J-C'':^ 

J      A.- 

4              X 

t              ^ 

5                               V 

^                  t 

\                     _l 

IT  11  1  n-+==HPHf-f  1  t-n  1  ni  T 

v^L  -^'^^ 

J~'\            /                      ^           -=^^v 

—  '     '^~^                            '\—'^           '^^'y 

±'~ ■^—■^'^A 

\        -"^                                ' 

'^-^            ^' —^-^^  ^\^^  .^'^ 

---' 

A.  Sunlight. 

B.  Temperature. 

C.  Saturation  deficit. 

D.  Wind  velocity. 


E.  Evaporation, 

blotting  paper 
atmomet«r. 

F.  Evaporation, 

porous  cup 
atmometer. 

G.  Absolute  humidity. 
H.  Barometric  pressure. 


I.  Transpiration  of 
potted  plant 
(Cow-beet) . 
K.  Transpiration  of 
cut  leaves  in 
potometers 
(Cow-beet) . 


L.  Relative  humidity. 
M.  Vapor  pressure 

deficit. 
N.  Dew-point  depression 


9    10    11    MT. 


2345     6     7     8910    11  NOON  1      2    3     4-56 


Fig.  32. — Series  33,  showing  evaporation,  transpiration,  and  the  factors  concerned 
for  September  8-9,  1919. 


62         EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 

The  evaporation  from  the  two  types  of  atmometers,  transpiration 
from  potometers  and  phytometers,  relative  humidity,  absolute 
humidity,  saturation  deficit  of  the  air,  vapor-pressure  deficit,  dew- 
point  depression,  barometer  pressure,  wind  velocity,  air-temperature, 
and  sunlight  are  shown  in  figure  32.  These  data  were  taken  from 
series  33  started  at  6  p.  m.  September  8,  and  ended  7  p.  m.  September 
9,  1919.  The  evaporation  from  the  two  types  of  atmometers  does 
not  agree,  and  a  study  of  the  maxima  and  minima  of  the  curves  shows 
that  the  Livingston  white  cyHnder  is  much  more  responsive  to  wind 


.2 
3.0 
JB 
.6 
.4 
.2 
2.0 
6 
j6 
.4 
.2 
I.O 
£ 
.6 
A 
.2 
0.0 


JL 

4 

□ 

A            1 

f-^+  JT 

-t   ^4    4T 

4^       A      t      \r 

t     Xt  I 

t        U     ^ 

t        V 

d- 

ii 

1 

I                                4 

ft                                t 

IP                                t 

i                        r           JL 

4    ^                                  t                 .[ 

it                                       La                  - 

^    t                                    ifc^:^ 

t    r-^                        ^t       ^^        - 

X  ^^                   4t       I 

^2       t                      jt          3 

\            jt 

\            4            f  J 

A      ^^->^    i         I 

^w         ^^  i         4 

A      -7   ^^        •->    ^^4              ^-   ■- 

^v    2      ^1-    ^    \      4                 I-    - 

^-                     ^V           ^^^7^                              1        + 

10    II   MT.  •     2    3    4    5     6     7     8    9    K)    M  NOON  I     2    3    4    5    6 


FiQ.  33. — Series  33,  showing  evaporation  from  white-cylinder  porous  cup  in 
cubic  centimeters  per  hour  (A),  compared  with  product  of  vapor- 
pressure  deficit  and  wind  velocity  (B),  calculated  by  Johnston's 
method. 

and  much  less  to  sunlight  than  the  blotting-paper  type.  Neither 
type  of  atmometer  showed  any  correlation  whatever  between  its 
water-loss  and  transpiration  from  potted  plants  or  cut  leaves.  The 
lack  of  any  relationship  in  the  curves  representing  water-loss  from 
potometers  and  phytometers  shows  the  entire  unreliability  of  cut 


EVAPORATION   AND    CAUSAL   FACTORS.  63 

stems  in  determining  the  normal  hourly  transpiration  from  rooted 
plants.  The  data  from  the  various  series  were  used  to  calculate  the 
theoretical  water-loss  of  the  white-cylinder  atmometers  according 
to  the  method  employed  by  Johnston  (1919),  following  the  precaution 
of  averaging  the  vapor-pressure  deficit  for  the  beginning  and  end  of 
each  hour  as  prescribed.  The  results  calculated  in  this  manner  for 
series  33  with  those  actually  obtained  with  the  atmometer  are  shown 
graphically  in  figure  33.  As  the  curves  show,  the  relation  is  not  at 
all  close.  But  when  the  wind  velocity  is  divided  by  a  factor  to 
reduce  its  effect  upon  the  results,  and  a  constant  added  to  represent 
the  replacement  by  diffusion  of  still  air  around  the  evaporating 
surface,  the  calculated  results  are  brought  into  much  closer  accord 
with  the  observed  rate  of  evaporation.  Even  then  there  are  discrep- 
ancies that  can  not  be  explained  fully  by  radiant  energy,  as  sug- 
gested by  Johnston.  The  method  may  be  of  value,  however,  as  a 
first  step  toward  an  analysis  of  the  part  played  by  each  factor  in 
producing  water-loss  from  the  atmometer,  and  thus  lead  the  way  to 
a  new  attack  upon  the  problem  of  transpiration. 

In  all  the  series  made  in  the  Great  Salt  Lake  region,  low  relative 
humidity  and  strong  sunlight  were  found  associated,  and  usually 
higher  temperatures  as  well.  For  this  reason  it  v/as  not  easy  to 
separate  the  action  of  each,  but  a  study  of  the  precise  effect  of  each 
was  beyond  the  scope  of  the  investigation.  The  general  effect  of 
increased  evaporation  as  determined  by  these  three  factors  is  shown 
for  alfalfa  in  figure  9.  This  represents  essentially  what  occurs  in  most 
of  the  mesophytes  studied,  low  relative  humidity  and  intense  sun- 
light producing  day  closure.  On  the  other  hand,  no  closure  was  found 
taking  place  during  the  day  if  the  humidity  was  high.  If  the  water- 
content  had  become  critical,  no  opening  occurred  at  all,  even  under 
a  condition  of  high  humidity,  but  if  the  plant  could  obtain  sufficient 
moisture  to  produce  opening,  the  stomata  would  show  no  mid-day 
closure.  The  increased  irregularity  in  the  rate  of  movement  following 
increased  evaporation  is  shown  in  figure  9. 

Each  factor  concerned  plays  at  times  a  role  independent  of  evapora- 
tion in  producing  changes  of  stomatal  movement.  Thus,  light  which 
acts  through  radiant  energy  in  producing  water-loss  from  the  plant 
has  been  shown  to  act  independently  of  air-temperature.  The  latter, 
which  is  a  very  important  factor  in  evaporation,  also  plays  an 
important  part  in  the  speed  at  which  opening  occurs.  Humidity  is 
more  closely  related  to  evaporation,  but,  when  high,  precipitation 
occurs  in  the  form  of  dew  or  rain,  and  this  causes  changes  in  stomatal 
movement.  Wind  increases  the  effect  of  low  humidity,  but  also 
carries  dust,  and  when  high  probably  also  causes  currents  of  air  to 
flow  through  the  leaf. 

The  effect  produced  upon  stomatal  movement  by  the  wetting  of 
the  leaves  by  dew  or  rain  is  most  clearly  shown  in  series  35,  begun 


64 


EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 


at  5  a.  m.  September  19,  and  ended  at  C  a.  m.  September  20,  1919. 
A  heavy  dew  had  fallen  during  the  night  before  and  the  leaves  were 
wet,  the  upper  surfaces  much  more  than  the  lower.  The  humidity 
was  very  high  during  the  series,  being  98  per  cent  at  the  start,  de- 
creasing slowly  to  a  minimum  of  49  per  cent  at  3''  40™  p.  m.  and 
increasing  again  to  80  per  cent  or  more  during  the  following  night. 
The  day  was  only  moderately  warm,  and,  while  there  were  no  clouds, 
the  large  amount  of  water-vapor  and  the  time  of  the  year  prevented 
sunlight  from  approaching  maximum.  The  stomata  of  both  sur- 
faces w^ere  closed  at  the  outset,  but  the  upper  opened  10  per  cent  at 

6  a.  m.  and  30  per  cent  the  following  hour.  At  this  time  the  lower 
stomata,  which  had  been  closed,  opened  50  per  cent.  By  8  a.  m. 
the  dew  had  dried  on  the  lower  surfaces  of  the  leaves  and  in  response 
the  stomata  closed  to  20  per  cent.  They  then  began  to  reopen,  the 
maximum  occurring  at  1  p.  m.  The  stomata  of  the  upper  surface 
continued  opening  rather  uniformly  until  9  a.  m.,  when  they  reached 
90  per  cent.  In  response  to  evaporation  of  the  heavy  dew  upon  the 
surfaces,  the  stomata  closed  to  70  per  cent  the  next  hour.  Maximum 
opening  was  reached,  however,  at  11  a.  m.  The  stomata  of  both 
surfaces  remained  wdde  open  until  5  p.  m.,  when  they  started  to  close, 
the  lower  closing  only  slightly  more  rapidly  than  the  upper.  Closure 
was  complete  in  the  lower  stomata  and  nearly  so  in  the  upper  at 

7  p.  m.    At  this  time  dew  again  began  to  form,  and  at  8  p.  m.  was 

100 

90 
80 
70 
60 


^ 

j 

"7 

^ 

y 

1 

1 

\ 

E 

■ 

•••■ 



... 
•• 

\ 

i 

/ 

=>r 

. 

u 

.. 

■■■' 

'^ 

A 

N 

7 

\ 

^i 

L, 

y 

B/ 

\ 

\ 

^ 

»■' 

\ 

"^^ 

^ 

0 

1 

' 

V 

/ 

\ 

\ 

■*• 

-- t--i._^ 

k— 

-- 

/ 

h 

/ 

/ 

\ 

\ 

~^ 

// 

'> 

^'/ 

^ 

\ 

\ 

A 

^ 

/ 

/ 

\ 

^ 

^ 

\ 

/ 

^ 

\ 

/ 

f  ^ 

/ 

L 

X 

A-- 

^^ 

Js 

z. 

_\ 

1 — 

7      8      9      10 


NOON  I       2      3 


5      6      7      8      9      10     II     MT. 


2      3      A      5      6 


FiQ.  34. — Series  35,  showing  stomatal  movement  in  heavily  watered  plants  of 
alfalfa,  the  partial  closure  at  8  and  10  a.  m.  following  disappearance 
of  dew;  upper  stomata  (A),  lower  stomata  (B),  sunlight  (C),  tempera- 
ture (D),  humidity  (E). 

distinctly  noticeable  on  the  upper  surfaces  of  the  leaves.  In  conse- 
quence, the  upper  stomata  began  to  reopen  very  slowly,  reaching 
25  per  cent  at  1  a.  m.  At  this  time  a  breeze  sprang  up,  causing  closure 
the  next  hour,  but  this  died  away  and  the  stomata  again  opened, 
reaching  20  per  cent  at  4  a.  m.  The  next  hour  they  closed,  and  re- 
mained in  this  condition  until  6  a.  m.,  when  the  series  ended.  The 
lower  stomata  were  closed  all  night,  except  for  5  per  cent  opening 
at  11  p.  m.  and  midnight,  when  the  dew  first  affected  the  lower  sur- 
faces (fig.  34).    When  the  leaves  and  stems  of  alfalfa,  sweet  clover, 


EVAPORATION   AND    CAUSAL   FACTORS. 


65 


and  nasturtium  were  wetted  experimentally  the  stomata  opened. 
Closure,  however,  did  not  always  follow  the  drying  of  this  moisture 
as  rapidly  as  it  did  in  this  series. 

Wind  carries  fine  particles  of  dust  to  coat  the  leaves,  thus  reducing 
light  penetration  and  chlorovaporization  and  indirectly  affecting 
stomatal  movement.  Dust  particles  are  also  found  wedging  open 
many  of  the  stomata  after  a  violent  wind.  A  stoma  of  wheat  kept 
from  closing  in  this  manner  is  shown  in  plate  1.  In  one  leaf  of  sugar- 
beet,  17  per  cent  of  all  the  stomata  observed  were  wedged  open  by 
dust  particles.  This  is  an  unusually  large  proportion,  but  at  many 
times  sufficient  stomata  in  each  leaf  are  found  permanently  held  open 
in  this  manner  to  produce  a  serious  effect  upon  the  plant  during 
times  of  stress.  High  winds  which  arise  suddenly  seem  to  force 
currents  of  air  through  the  leaves  before  the  stomata  close,  and 
wedge  the  grains  into  the  slit  in  this  manner.  This  is  also  the  most 
plausible  explanation  of  the  immediate  great  increase  of  transpiration 
which  occurs  at  once  upon  the  sudden  appearance  of  a  high  wind. 
Under  ordinary  circumstances,  however,  wind  probably  only  removes 
shells  of  nearly  saturated  air  from  the  outer  ends  of  the  stomatal 
pores  (Dixon,  1914). 

The  attempt  to  correlate  evaporation  from  atmometers  and  free 
water-surfaces  with  transpiration  has  met  only  with  negative  results. 
A  porous  cup  can  hardly  be  expected  to  respond  more  freely  to  its 
environment  than  a  free  water-surface  or  saturated  blotting  paper. 


90 
80 

~" 

A 

/ 

\ 

^ 

^ 

/" 

_ 

— 

^\ 

^ 

70 
60 
50 
AO 
30 
?0 
10 

\ 

V 

r" 

,-■> 

/ 

\ 

^ 

/ 

^ 

y 

^ 

[/ 

'\ 

--. 

N, 

/ 

p 

^ 

/ 

/ 

/ 

^ 

N 

v^ 

B 

\ 

-^ 

N 

^ 

/^ 

' 

/ 

V 

,•- 

\ 

~~ 

"•- 

— 

—  H 

-- 

> 

y 

J 

/ 

\ 

A 

\ 

/ 

^ 

^ 

1 ] 

/ 

\ 

0 

\ 

5       6       7      8      9      10 


NOON  1       2      3 


Fig.  35.- 


" Relative  transpiration"  based  on  evaporation  from  blotting-paper 
atmometer  (A)  and  from  white-cylinder  porous  cup  (B),  compared 
with  stomatal  movement  in  onion  (C). 


and  hence  the  results  from  a  white-cylinder  atmometer  have  no 
advantages  over  those  from  the  other  types.  Until  an  atmometer  is 
devised  which  responds  in  the  same  manner  and  degree  to  each  of  the 
factors  concerned,  the  ratio  of  transpiration  to  evaporation  is  mean- 
ingless. Thus,  in  series  34,  ''relative  transpiration,"  calculated  from 
the  data  obtained  by  the  blotting-paper  atmometer,  shows  a  certain 
degree  of  correlation  with  stomatal  movement,  while  that  calculated 
from  the  evaporation  from  the  porous  cup  shows  practically  none 


66    EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 

(fig.  35).  There  is,  in  consequence,  no  warrant  for  the  view  that  lack 
of  agreement  be  ween  stomatal  movement  and  "relative  transpira- 
tion" based  upon  evaporation  from  the  porous-cup  atmometer 
indicates  that  stomata  are  non-regulatory.  Nor,  on  the  other  hand, 
can  the  lack  of  close  agreement  between  * 'relative  transpiration" 
based  upon  the  blotting-paper  type  of  atmometer  be  attributed  to 
incomplete  regulation  by  the  stomata  with  as  much  justice  as  that 
this  evaporimeter  fails  to  respond  in  the  same  manner  and  degree 
to  the  various  factors  as  the  plant,  aside  from  the  effect  of  stomatal 
movement.  The  only  conclusion  permissible  is  that  the  blotting- 
paper  atmometer  represents  with  somewhat  greater  fidelity  the 
water-loss  from  the  plant  when  it  responds  freely  to  its  environment 
than  does  the  porous  cup. 

WATER-CONTENT. 

Water-content  acts  upon  stomatal  movement  by  changing  the  rate 
at  which  water  is  supplied  to  the  leaves.  When  humidity  is  high, 
therefore,  a  moderately  low  water-content  may  not  produce  any 
significant  difference  in  movement  as  compared  with  that  found  in  a 
plot  of  highly  watered  plants.  But  if  humidity  becomes  low,  tem- 
perature high,  and  sunlight  intense,  the  same  water-content  may  be 
critical  and  produce  striking  differences  in  movement.  The  Great 
Salt  Lake  region  was  peculiarly  adapted  to  studies  dealing  with  the 
divergence  of  movement  caused  by  differences  of  water-content  in 
relation  to  evaporation,  as  the  rainfall  is  deficient,  permitting  the 
water-content  to  be  readily  controlled,  and  very  humid  days  are 
found  as  well  as  very  dry  ones.  Attempts  to  conduct  similar  experi- 
ments at  the  greenhouse  of  the  University  of  Minnesota  failed  to 
show  anything  conclusively,  since  the  humidity  could  not  be  lowered 
materially,  or  the  much  greater  changes  produced  by  deficient  light 
be  discounted.  Hence,  to  be  conclusive,  such  experiments  must  be 
conducted  in  the  field  in  a  dry,  irrigated  region. 

Four  plots  of  alfalfa  plants  were  used  in  series  32,  started  at  7  p.  m. 
August  25  and  ended  7  p.  m.  August  26,  1919.  The  plants  of  the 
first  plot  were  water-logged;  the  second  was  irrigated  with  2  inches 
of  water  daily,  beginning  a  week  before  the  start  of  the  series;  the 
third  had  been  irrigated  the  week  before  with  4  inches  of  water; 
and  the  fourth  had  not  been  irrigated  for  more  than  a  month.  The 
soil  of  plot  1  was  saturated,  plot  2  had  a  water-content  of  32  per  cent 
at  the  start  of  the  series,  plot  3  had  24.3  per  cent,  and  plot  4  had  10.6 
per  cent.  The  plants  of  plot  1  presented  a  wilted  appearance  through- 
out the  series,  those  of  plots  2  and  3  were  normal,  while  those  of  plot 
4  wilted  somewhat  during  the  day  and  recovered  at  night.  The 
weather  conditions  during  the  series  were  rather  favorable,  although 
the  temperature  rose  to  89°  F.  at  2  p.  m.  August  26.  A  haze  formed 
after  sunrise  and  thickened  into  light  clouds  at  10  a.  m.,  which  floated 


WATER-CONTENT. 


67 


away  and  disappeared  by  1  p.  m.  At  2,  3,  and  5  p.  m.  passing  clouds 
reduced  the  light  for  short  periods  of  time.  The  humidity  was  not 
extremely  low,  as  often  happened  in  the  region,  since  the  minimum 
was  25  per  cent  at  3  p.  m.,  while  the  average  for  the  day  was  over 
35  per  cent.  The  wind  was  slight,  except  between  8  and  9  a.  m.,  when 
it  rose  to  2.16  miles  per  hour. 

The  stomata  of  the  plants  of  plot  1  were  closed  throughout  the 
series,  as  was  expected  from  the  appearance  of  the  plants.  Those 
of  the  plants  in  plot  3  were  17  per  cent  open  at  the  start  and  closed 
completely  by  9  p.  m.  They  remained  closed  until  4  a.  m.,  when  the 
first  light  of  dawn  caused  them  to  open  slightly,  reaching  10  per  cent 
at  5  a.  m.  Sunrise  then  caused  them  to  open  smoothly  and  uniformly 
to  maximum  at  8  a.  m.,  in  which  condition  they  remained  until  4 
p.  m.,  when  they  started  to  close.  They  had  decreased  only  to  85 
per  cent  at  6  p.  m.,  but  the  following  hour  they  closed  to  20  per  cent. 
The  stomata  of  plot  2,  which  had  nearly  saturated  soil,  showed 
stomata  open  from  10  to  25  per  cent  throughout  the  night,  but 
closing  completely  at  4  a.  m.  The  following  hour  they  opened  20 
per  cent  and  then  more  rapidly  until  maximum  was  reached  at  7 
a.  m.  They  remained  wide  open  until  2  p.  m.,  when,  as  usual,  the 
plot  was  given  2  inches  of  water.  In  consequence,  they  commenced 
closing  immediately  and  smoothly  to  60  per  cent  at  4  p.  m.  By  this 
time  the  water  had  soaked  into  the  ground  and  the  upper  surface 
was  drying  slightly.    The  stomata  remained  at  60  per  cent  for  an 


MMIlilllllli 


7      8     9      10     II     MT. 


234.56789     10     II  NOON  I      2      3 


FiQ.  36. — Effect  of  water-content  on  movement  in  upper  stomata  of  alfalfa ; 
irrigated  daily  with  2  in.  of  water  (A),  irrigated  7  days  before 
with  4  in.  (B),  not  irrigated  (C). 

hour  and  then  opened  to  80  per  cent  at  6  p.  m.  The  next  hour  they 
closed  completely.  The  stomata  of  the  plants  of  plot  4  were  closed 
at  the  start  of  the  series  and  remained  closed  until  10  p.  m.  Then 
they  opened  very  slowly,  reaching  9  per  cent  at  1  a.  m.,  thereafter 
more  rapidly  and  irregularly  to  28  per  cent  at  5  a.  m.  As  a  result  of 
daylight,  they  opened  to  45  per  cent  at  6  and  70  per  cent  at  7  a.  m. 
The  next  hour  they  closed  and  remained  closed  until  the  end  of  the 
series,  except  for  slight  irregular  openings  characteristic  of  plants 
operating  with  deficient  leaf -water  (fig.  36). 


68    EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 

The  movement  shown  by  the  plants  of  plot  3  is  characteristic 
of  the  normal  light-induced  behavior  of  alfalfa  stomata  under  favor- 
able conditions,  save  in  one  very  minor  point ;  the  slight  irregularity 
in  maintaining  maximum  opening,  especially  toward  the  last,  shows 
that  evaporation  was  just  on  the  verge  of  becoming  critical.  The 
higher  water-content  of  plot  2  caused  the  stomata  to  show  partial 
opening  throughout  the  night.  This  is  not  characteristic  of  stomatal 
movement  of  the  alfalfa  group  of  plants  under  normal  conditions,  as 
found  in  the  regions  in  which  these  experiments  were  conducted. 
It  may  be  characteristic  of  such  plants,  however,  in  regions  where 
there  is  a  heavy  rainfall  each  day.  Maximum  opening  was  reached 
by  the  plants  of  this  plot  an  hour  earlier  than  those  of  plot  3.  The 
closure  induced  by  irrigation  may  be  attributed  either  to  the  cutting 
off  of  air  from  the  roots,  which  seems  improbable,  or  to  chilling  or 
shock  inhibiting  their  functioning  for  a  time.  The  wilting  and  con- 
tinued closure  of  stomata  of  the  plants  of  the  first  plot,  however, 
was  undoubtedly  due  to  the  cutting  off  of  air  to  the  roots,  causing  them 
to  fail  to  function. 

The  stomata  of  the  plants  in  the  fourth  plot  showed  the  effect  of 
very  low  water-content.  The  plants  began  to  recover  turgor  after 
sundown,  and  the  first  effect  was  observable  at  11  p.  m.,  when  slight 
opening  of  the  stomata  occurred.  Not  until  after  1  a.  m.,  however, 
was  the  water  lost  during  the  day  fully  replaced  in  the  leaves.  With 
the  appearance  of  daylight  the  stomata  of  these  plants  began  to 
open,  as  well  as  those  of  the  other  two  plots,  but  much  more  slowly. 
At  7  a.  m.  the  water-loss  was  becoming  critical  and  the  following 
hour  the  stomata  closed.  The  water-content  of  this  plot  was  so  low 
that  recovery  of  turgor  was  incomplete,  as  well  as  very  much  delayed. 
As  shown  in  figure  9,  when  evaporation  is  great  and  water-content 
only  moderately  low,  night  opening  occurs  much  earlier  and  is  con- 
siderably greater. 

LEAF  TURGOR. 

The  amount  of  water  in  leaves  normally  changes  throughout 
a  24-hour  period.  The  changes  are  slight  when  water-content  is 
high  and  evaporation  low,  but  they  are  usually  great  with  mod- 
erate water-content  and  high  evaporation.  The  percentage  of 
water  present  in  the  leaf  at  any  time  is  determined  by  the  rate  of 
transpiration  on  the  one  hand  and  the  rate  of  water-supply  on  the 
other.  When  the  stomata  open  at  daylight  and  the  factors  con- 
cerned in  evaporation  become  more  intense,  the  rate  of  water- 
supply  soon  falls  behind  that  of  transpiration.  In  consequence, 
the  amount  of  water  in  the  leaf  begins  to  decrease,  until  the  loss 
often  becomes  critical.  At  this  time  the  stomata  close  partially  or 
completely,  and  transpiration  is  diminished.  If  this  reduces  the 
rate  of  water-loss  sufficiently,  the  leaves  begin  to  regain  turgor,  and 


LEAF   TURGOR.  69 

after  a  time  the  stomata  reopen;  otherwise,  they  remain  closed 
until  nightfall  brings  on  a  decrease  in  the  intensity  of  the  factors 
producing  evaporation,  and  the  turgor  again  rises. 

A  certain  amount  of  the  water  present  in  a  turgid  leaf  may  be 
regarded  as  the  working  margin.  The  loss  of  part  of  this  margin 
does  not  seem  to  interfere  in  any  manner  with  the  functioning  of 
the  leaf,  and  it  is  probable,  even  when  it  is  wholly  lost  and  the 
stomata  are  closing,  that  photosynthesis  and  other  functions  are 
still  carried  on,  in  part  at  least.  Its  presence,  therefore,  permits  the 
stomata  to  remain  open  and  carbon-dioxid  absorption  to  go  on 
for  a  time,  in  spite  of  excessive  evaporation.  As  the  amount  of 
leaf-water  changes  not  only  during  a  24-hour  period  but  also  from 
one  rain  or  irrigation  to  another,  this  working  margin  also  changes. 
The  content  of  water  in  alfalfa  leaves  has  been  found  to  change  from 
a  maximum  of  410  per  cent  of  dry  weight  after  several  days  of  rain 
and  high  humidity  to  290  per  cent  of  the  dry  weight  when  on  the 
verge  of  wilting  as  a  result  of  low  water-content  a  month  later.  As 
the  critical  minimum  of  leaf-water  at  which  stomatal  closure  occurs 
varies  in  lesser  proportion,  there  must  be  some  means  of  adjustment 
to  decrease  in  water-content  by  reduction  of  the  amount  of  water 
with  which  a  leaf  can  operate. 

The  failure  to  recognize  the  presence  of  this  working  margin  of 
water  in  the  leaf  led  Lloyd  (1912)  and  Knight  (19176)  to  conclude 
that  the  stomata  had  no  active  part  in  keeping  up  leaf  turgor. 
Lloyd  concludes  from  his  experiment  that  '^  decrease  in  water  in  the 
leaf  occurs  during  the  opening  of  the  stomata.  These  organs  are, 
therefore,  not  closely  regulatory  of  the  loss  of  water  and  are  in- 
effectual in  maintaining  a  constant  supply  of  leaf- water."  Knight 
states  that  ''the  experiments  in  the  present  paper  have  confirmed 
Lloyd's  results,"  and  later  that  ''the  stomatal  aperture  is  not  re- 
duced by  slight  water  deficiency  in  the  leaf;  hence  the  ordinary 
view  that  the  stomata,  by  their  response  to  incipient  drying,  are 
the  chief  factors  in  maintaining  the  water-content  of  the  leaf  is  not 
tenable.  On  the  other  hand,  the  stomata  are  very  sensitive  to  light 
changes,  so  that  with  increasing  Hght  intensity  the  stomata  may  con- 
tinue to  open,  whilst  the  water-content  of  the  leaf  is  decreasing." 

The  assumptions  made  by  both  writers  seem  to  be  that  if  it 
were  not  for  the  ineffectiveness  of  the  stomata,  the  water  present 
in  the  leaves  would  be  constant,  and  that  any  decrease  in  the  water 
contained  in  the  leaf  inhibits  its  normal  functioning.  The  first 
assumption  would  then  mean  that  the  normal  leaf  water-content  is 
constant,  and  that  the  same  per  cent  of  water  is  found  night  after 
night,  and  departure  from  this  occurs  only  as  a  result  of  excessive 
transpiration  during  the  day.  This  has  not  been  found  to  be  the 
case,  however,  as  the  nightly  maximum  fluctuates  greatly  from  one 
rain  or  irrigation  to  another.     The  second  assumption  would  mean 


70         EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 

that  the  plot  of  alfalfa  plants,  for  instance,  having  the  highest 
average  of  water  in  their  leaves  during  each  24-hour  period  would 
exhibit  the  greatest  growth  and  would  produce  the  most  dry  matter. 
This  again  has  not  been  found  to  be  the  case.     The  plants  of  plot 

2  used  in  series  32,  which  was  discussed  in  connection  with  water- 
content,  showed  a  higher  average  per  cent  of  leaf-water  than  did 
the  plants  of  plot  3,  but  the  plants  of  the  latter  showed  the  greatest 
increase  of  dry  matter  during  the  two  weeks'  observation.  The 
greater  decrease  of  leaf-water  during  the  day  of  the  plants  in  plot 

3  can  not  be  assumed  to  have  interfered  in  any  manner  with  their 
functioning.  Hence,  it  may  safely  be  said  that  fluctuations  of 
leaf -water  are  normal  and  universal.  When  not  so  great  as  to 
induce  closure  of  the  stomata,  they  do  not  inhibit  or  in  any  other 
manner  affect  photosynthesis,  translocation,  or  other  functions  of 
the  leaf.  Such  fluctuation  may  be  said  to  occur  only  in  the  working 
margin  of  the  leaf-water. 

Changes  of  leaf  turgor  offer  the  best  explanation  regarding  the 
mechanism  of  mid-day  closure  and  night  opening.  It  has  always 
been  found  that  mid-day  closure  occurs  when  the  leaf-water  has 
been  reduced  to  a  point  which  is  the  safe  minimum  for  a  given  water- 
content.  The  stomata  do  not  reopen  until  the  per  cent  of  water 
rises  once  more  above  this  point  and  the  leaf  again  has  a  margin 
with  which  to  safely  operate.  It  is  not  known  definitely  whether 
this  is  due  to  the  effect  of  loss  of  turgor  causing  reconversion  of 
starch  and  thus  producing  closure,  or  whether  evaporation  concen- 
trates the  sap  in  the  adjacent  cells  to  a  sufficient  degree  to  cause 
exosmosis  from  the  guard-cells.  Inspection  of  the  relation  between 
the  starch  index  curve  and  stomatal  curve  for  series  10  (fig.  29) 
would  indicate  that  both  are  possibly  involved.  Night  opening 
occurs  only  in  those  leaves  in  which  turgor  is  recovered  faster  than 
starch  is  stored  in  the  guard-cells.  It  therefore  seems  plausible 
that  the  sap  of  the  parenchymatous  and  adjacent  epidermal  cells  is 
diluted  more  rapidly  by  this  increase  of  turgor  than  that  of  the 
guard-cells  is  by  the  removal  of  sugar.  This  would  then  produce 
a  relatively  higher  concentration  of  sap  in  the  guard-cells  and  con- 
sequent opening  of  stomata,  in  spite  of  the  fact  that  such  concen- 
tration was  decreasing.  The  great  difference  found  by  Iljin  (1914) 
between  the  concentration  of  sap  in  the  guard-cells  and  the  sur- 
rounding epidermal  cells  would  make  it  impossible  for  night  opening 
to  occur  in  this  manner,  but  his  results  are  undoubtedly  too  high. 
Wiggins  (1921),  repeating  this  work  with  apparently  much  greater 
care,  found  considerably  less  difference.  Because  he  failed  to  disrupt 
the  neighboring  epidermal  cells,  however,  the  solutions  producing  plas- 
molysis  of  the  guard-cells  were  probably  diluted  by  passing  through 
these  adjacent  cells,  and  hence  his  findings  are  also  too  high.  It  seems 
hardly  possible,  for  example,  that  the  guard-cells  can  lose  their  turgor, 


PLANT   HABIT   AND    CONDITION.  71 

and  cause  closure  while  they  still  have  a  distinctly  higher  concentra- 
tion than  the  adjacent  cells.  The  solution  of  this  problem  therefore 
requires  further  investigation. 

PLANT  HABIT  AND  CONDITION. 

The  growth  habit  of  a  plant  influences  the  stomatal  movement  by 
introducing  differences  in  the  relation  of  the  rest  of  the  plant  to 
the  stomata.  Thus,  the  trees  investigated  did  not  exhibit  mid-day 
closure,  even  on  days  when  this  was  extreme  in  all  the  herbs  studied. 
The  greater  depth  to  which  tree  roots  penetrate  would  permit  these 
to  draw  upon  supplies  of  moisture  not  available  to  many  other 
plants.  This,  however,  does  not  explain  why  the  Lombardy  poplar 
showed  no  mid-day  closure  on  a  day  when  alfalfa  exhibited  extended 
and  complete  mid-day  closure,  since  the  roots  of  both  plants  reached 
the  moist  soil  just  above  the  water-table.  As  this  type  of  behavior 
was  found  in  all  the  trees  studied,  it  is  undoubtedly  the  result  of 
their  growth  habit  and  may  be  attributed  to  the  great  amount  of 
water  present  in  the  trunk,  which  acts  as  a  reserve.  Dendrograph 
studies  carried  on  with  Pseudotsuga  taxifolia  and  Pinus  ponderosa 
at  the  Alpine  Laboratory  show  that  the  water  present  in  the  trunks 
probably  moves  with  great  rapidity  up  to  the  leaves. 

Stomatal  movement  in  herbaceous  plants  is  subject  to  great 
variation  as  a  rule,  because  the  leaf  has  no  reserve  of  water  to  draw 
upon.  Hence,  when  the  working  margin  has  disappeared,  it  is 
necessary  for  such  a  leaf  to  restrict  water-loss  and  reduce  its  functions 
accordingly.  The  thin-leaved  plants  naturally  have  a  smaller 
margin  of  water  than  the  thicker-leaved  ones,  and  hence  show 
earlier  and  more  extended  closure  as  a  rule.  With  a  moderate 
water-content  such  leaves  may  show  several  periods  of  day  closure 
under  extreme  conditions  of  evaporation.  Fleshy-leaved  forms,  on 
the  other  hand,  rarely  show  more  than  one,  and  mid-day  closure  is 
accompanied  by  visible  wilting  in  all  the  cases  studied.  All  of  the 
plants  in  the  group  with  normally  open  stomata  at  night  under 
optimum  conditions  are  rather  thick-leaved.  As  the  stomata  of 
alfalfa  opened  at  night  after  normal  day  opening  only  when  the 
water-content  was  abnormally  high,  night  opening  generally  is  due 
probably  to  a  greater  available  supply  of  water  in  the  thicker  leaves. 
Some  of  the  shrubs  showed  the  same  kind  of  behavior  as  the  trees, 
but  others  exhibited  some  degree  of  mid-day  closure  at  times,  and  are 
intermediate  in  behavior  between  trees  and  herbs. 

The  age  or  degree  of  maturity,  as  well  as  the  growth  habit  of  a 
plant,  affects  its  stomatal  movement.  Young  cabbage  plants  showed 
greater  and  longer  continued  opening  during  the  day  than  plants 
ready  to  head.  Ripening  barley  showed  no  movement  at  the  time 
when  plants  started  later  and  not  yet  headed  out  showed  4  hours 
opening  during  the  morning.     In  series  32,  two  other  plots  were 


72         EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 

included  with  essentially  the  same  water-content  as  plot  3,  but  in 
different  stages  of  growth.  In  plot  6,  the  plants  were  0.8  dm. 
high,  in  7,  2.0  to  2.5  dm.  high,  and  in  plot  3  they  had  reached  a 
height  of  3.5  to  4.5  dm.  and  were  beginning  to  flower.  The  dif- 
ferences found  in  stomatal  movement  were  insignificant  and  readily 
explained  bj^  the  slight  differences  in  water-content  and  error  of 
measurement.  In  series  34  the  experiment  was  repeated.  The 
new  growth  of  the  first  plot  averaged  0.7  dm.,  the  second,  2.0  dm. 
and  in  the  third  plants  were  just  starting  to  blossom.  As  the  water- 
content  was  lower  and  evaporation  higher,  2  hours  of  mid-day 
closure  was  found  in  the  second  plot,  none  in  the  first,  and  slight 
closure  in  the  third.  The  greater  proportion  of  roots  to  leaf  area  in 
the  first  plot  explains  its  lack  of  closure,  but  the  reason  for  the 
greater  opening  in  the  blossoming  plants  as  compared  with  the 
half-grown  plants  is  not  so  clear. 

The  age  of  the  leaf  also  affects  its  stomatal  movement.  This  was 
investigated  in  alfalfa,  barley,  corn,  Rumex  patientia,  sweet  clover, 
turnip,  potato,  and  sugar-beet.  In  all  these  the  stomata  are  the 
last  of  the  epidermal  cells,  and  probably  of  all  the  cells  in  the  leaf, 
to  be  formed.  With  most  of  the  plants  they  are  formed  at  nearly 
the  same  time,  potato  being  the  one  conspicuous  exception.  In 
consequence,  they  also  begin  to  function  at  the  same  time,  a  few 
starting  a  day  or  two  earlier  than  the  others.  At  first,  opening  is 
slight  and  very  brief,  but  after  a  few  days  it  becomes  more  pro- 
longed. However,  a  certain  number  are  badly  formed,  resulting  in 
mechanically  or  physiologically  defective  stomata  which  never  open. 
About  the  time  that  the  stomata  approach  maturity  the  granular 
protoplasm  of  the  epidermal  cells  begins  to  disappear,  and  at  matu- 
rity these  are  rather  transparent  and  clear.  The  length  of  Ufe  of  a 
mature  leaf  depends  upon  the  species  of  the  plant  and  the  treat- 
ment to  which  it  is  subjected.  Bruising  by  wind,  injury  by  insects 
and  fungi,  exposure  to  extreme  evaporation  when  the  water-supply 
is  deficient,  drying  out  because  of  stomata  wedged  open  by  dust 
grains  and  other  vicissitudes,  often  shorten  its  life.  The  larger 
number  wither  in  the  natural  course  of  growth,  the  first  visible 
indication  of  which  is  a  slight  yellowing.  But  it  is  usually  unsafe 
to  strip  the  next  younger  leaf  on  the  stem,  since  its  stomata  have 
begun  to  function  poorly  and,  before  any  very  evident  change  of 
color  occurs,  they  have  closed  permanently. 

Other  conditions  in  the  leaf  affect  stonmtal  movement.  The 
relation  of  the  stomata  of  the  two  surfaces  to  the  water-supply 
affects  this  movement,  as  shown  in  connection  with  sugar-beet. 
The  number  of  stomata  on  the  leaf  and  their  size  is  a  result  of  the 
degree  of  expansion  of  the  leaf,  which  in  turn  was  determined  in 
part  by  weather  conditions  during  development.  Thus,  a  leaf  of 
Malva  rotundifolia  had  241  stomata  per  square  millimeter  and 
averaging  6.1  m-  long,  when  developed  during  the  hot,  dry  weather 


PLANT   HABIT  AND    CONDITION.  73 

of  the  first  part  of  July  1916,  while  leaves  produced  a  month  earlier 
had  only  173  stomata  per  square  millimeter,  averaging  7.9  /x 
long.  The  ratio  of  stomata  to  other  epidermal  cells  was  the  same, 
and  hence  the  difference  was  merely  one  of  expansion  or  the  size 
of  the  cells.  This  expansion  does  not  affect  guard-cells  and 
epidermal  cells  equally,  since  the  average  increase  of  area  was  1.4 
greater  in  the  June  leaves  than  those  formed  in  July,  while  the  area 
of  each  pore  when  wide  open  was  1.7  greater. 

The  diffusive  capacity,  wva6^  of  the  stomata  when  wide  open 
was  very  nearly  the  same,  that  of  the  July  leaves  being  1,103  and 
the  June  leaves  966,  a  difference  of  less  than  5  per  cent.  As  this 
was  the  greatest  difference  found,  and  the  diffusion  capacity  of 
the  leaves  usually  checked  within  1.6  per  cent,  there  was  no  ad- 
vantage in  using  this  instead  of  the  percentage  of  maximum  opening, 
except  where  it  was  desired  to  compare  the  diffusion  capacity  of 
the  different  species  other  than  stomatal  movement.  Hence,  the 
difference  in  expansion  has  little  or  no  effect  upon  the  diffusion 
through  the  stomata,  or  upon  the  effect  of  stomatal  movement  upon 
such  diffusion.  The  stomata  of  these  larger  leaves,  however,  are  more 
sensitive  to  low  humidity  than  those  of  the  smaller  leaves.  It  may  be 
pointed  out  in  this  connection  that  Eckerson's  figures  for  the  number 
and  size  of  stomata  of  various  species  apply  only  to  greenhouse  plants, 
as  field  plants  differ  greatly  from  these  in  most  cases  examined. 

The  effect  of  hairs,  wax,  and  sunken  stomata  is  to  protect  the 
leaves  from  water-loss  to  a  certain  degree.  In  the  case  of  waxy 
leaves  and  leaves  with  sunken  stomata,  it  obviously  is  impossible 
to  determine  the  effect  of  these  by  direct  comparison,  but  in  the 
case  of  hairy  leaves  the  hairs  can  be  removed,  at  least  in  part.  Two 
experiments  were  performed  upon  Encelia  farinosa  in  March  1918, 
at  Tucson,  Arizona,  comparing  the  movement  in  "shaved"  and  in 
normal  leaves.  The  woolly  hairs  of  a  number  of  leaves  were  re- 
duced as  much  as  possible  with  a  safety  razor.  On  the  following 
diy  a  short  series  was  made,  beginning  at  8  a.  m.  and  ending  at 
noon.  The  stomata  of  the  shaved  leaves  were  wide  open  when  the 
series  started  and  remained  at  maximum  1  hour.  At  10  a.  m.  they 
had  closed  to  60  per  cent,  at  11  a.  m.  to  20  per  cent;  at  noon  closure 
was  complete.  The  stomata  of  the  normal  leaves  were  only  70 
per  cent  open  at  8  a.  m.,  opened  to  maximum  at  9  a.  m.,  and  re- 
mained in  this  condition  to  the  end  of  the  series.  The  second 
experiment  showed  essentially  similar  results.  The  removal  of 
most  of  the  hairs  increased  the  amount  of  light  reaching  the  stomata; 
this  accounts  for  the  earher  opening  in  the  shaved  leaves.  The 
closure  before  noon  may  be  ascribed  to  a  greater  water-loss  from 
the  leaves  when  the  hairs  were  removed.  Inspection  of  the  leaves 
supported  this  view,  but  unfortunately  no  determination  could  be 
made  of  the  percentage  of  water  in  the  shaved  and  normal  leaves 
at  the  time.     Nevertheless,  the  stomatal  closure  and  less  turgid 


74    EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 

appeareance  at  noon  of  the  shaved  leaves  is  evidence  that  hairs, 
especially  when  as  dense  as  on  the  leaves  of  this  plant,  are  of  con- 
siderable value  in  reducing  water-loss. 

Plants  adjust  and  adapt  themselves  to  their  environment  in 
a  number  of  ways,  one  of  which  is  stomatal  behavior.  The  hy- 
drophytes studied  had  permanently  open  stomata,  since  they  usually 
have  no  need  of  the  mobile  kind.  In  fact,  night  closure  would  prob- 
ably be  disadvantageous  to  a  plant  such  as  Scirpus  validus,  since  dif- 
fusion of  gases  through  the  air-passages  to  the  submerged  parts  must 
necessarily  be  slow  and  is  therefore  continued  throughout  the  night. 

Plants  which  grow  normally  in  a  very  humid  region,  where  the 
weather  rarely  becomes  hot  and  dry  and  the  water-content  low,  do 
not  show  the  rapid  response  to  evaporation  that  a  plant  developed 
in  a  more  arid  habitat  does.  Thus,  alfalfa  shows  a  greater  response 
to  low  humidity  than  potato  or  sugar-beet,  and  a  greater  response 
than  even  the  white-flowered  sweet  clover  {Melilotus  alba),  which 
has  very  similar  leaves  and  stomata.  The  plants  of  the  arid  regions 
have  generally  adapted  themselves  in  one  of  two  ways,  some 
functioning  at  all  times,  and  others  only  during  the  rainy  season. 
Opuntia  versicolor  belongs  to  the  former  class  (E.  B.  Shreve,  1916), 
its  stomata  functioning  normally  in  the  manner  that  alfalfa  stomata 
do  under  extreme  conditions.  Fouquiera  splendens,  on  the  other 
hand,  produces  a  crop  of  leaves  when  opportunity  permits,  which 
function  until  the  water-supply  runs  low,  in  the  same  manner  as 
a  mesophyte  under  optimum  conditions.  When  the  water-content 
runs  low  the  stomata  begin  to  show  less  and  less  opening,  until 
toward  the  last,  when  the  leaves  begin  to  change  color,  they  open 
very  little,  and  for  only  a  short  time  each  day.  This  behavior 
duplicates  that  of  an  aging  leaf  of  alfalfa.  Encelia  farinosa,  which 
tends  to  retain  some  leaves  at  all  times,  shows  behavior  much  like 
that  of  Fouquiera  when  conditions  are  favorable,  and  somewhat 
the  same  behavior  as  Opuntia  versicolor  when  these  are  unfavorable. 
Hence,  it  is  intermediate  between  the  two,  especially  since  it  drops 
most  of  its  leaves  upon  approach  of  unfavorable  conditions,  and 
those  that  remain  probably  show  opening  only  at  night,  if  at  all. 

SUMMARY. 

1.  Light  induces  opening  of  the  stomata  after  daybreak  by 
X  initiating  conversion  of  the  starch  within  the  guard-cells  into  sugar. 
This  increases  the  osmotic  pressure  of  the  guard-cells,  which  in 
turn  causes  the  increase  of  turgor  necessary  to  produce  opening. 
The  starch-content  of  the  guard-cells  never  wholly  disappears,  but 
usually  is  at  its  lowest  about  10  a.  m.  When  no  other  factor  influ- 
ences the  movement,  the  starch-content  rises  from  this  time  until 
just  before  daylight  the  next  morning.  During  the  middle  part  of 
the  day  and  until  shortly  before  the  stomata  start  to  close,  the  rise  in 
the  starch-content  is  very  slow,  but  is  rapid  during  closure.     After 


SUMMARY.  75 

closure  the  rate  of  increase  again  becomes  slow  and  is  further  re- 
tarded during  the  night. 

2.  Changes  of  movement  caused  by.  factors  other  than  light  are 
not  necessarily  accompanied  by  corresponding  changes  in  the  starch- 
content  of  the  guard-cells. 

3.  Reduction  of  light  to  less  than  half  of  normal  is  usually 
necessary  to  produce  any  effect  upon  the  stomata  of  plants  growing 
in  the  open.  When  the  stomata  are  closing,  they  respond  to  de- 
crease of  light  more  rapidly  than  when  opening. 

4.  Stomata  open  at  night  as  a  result  of  moonlight  or  a  strong 
artificial  light  of  much  less  intensity  than  1  per  cent  of  the  sunlight 
maximum.  They  open  more  readily  toward  morning  than  before 
midnight,  and  hence  at  night  also  the  reactions  which  tend  to  pro-- 
duce  opening  or  closure  are  more  readily  hastened  than  reversed. 

5.  The  temperature  of  the  air  affects  the  speed  at  which  the 
stomata  open  during  the  morning.  The  length  of  time  required 
for  opening  is  reduced  by  approximately  one-half  for  every  10°  C. 
rise  in  temperature.  This,  of  course,  occurs  only  within  the  limits 
of  temperature  at  which  protoplasm  functions. 

6.  When  the  temperature  of  the  soil  rises  too  much  the  stomata 
close,  and  in  extreme  cases  the  plant  wilts. 

7.  The  temperature  of  the  leaves  was  usually  found  to  be  lower 
than  that  of  the  air  when  the  stomata  were  open,  and  higher  when 
closed  in  sunlight. 

8.  A  high  humidity  of  the  air  permits  the  stomata  to  open 
wider  and  remain  open  longer  than  a  low  humidity  under  most 
conditions.  This  is  especially  true  when  the  water-content  has 
decreased  seriously  and  to  a  point  where  the  plant  has  great  diffi- 
culty in  obtaining  sufficient  water  to  meet  evaporation  during  the  day. 

9.  No  atmometer  or  evaporimeter  was  found  that  would  measure 
at  all  accurately  the  effect  upon  the  plant  of  all  the  factors  con- 
cerned in  evaporation. 

10.  When  the  leaves  of  a  plant  were  wet  by  dew  or  rain,  or  wet 
artificially,  the  stomata  usually  opened  if  closed,  or  opened  more 
widely  if  partially  open.  When  the  water  dried  the  stomata  closed 
wholly  or  partially. 

11.  Wind  caused  increases  of  transpiration  unlike  the  increases 
of  evaporation  as  measured  by  atmometers.  In  the  main,  the  plant 
showed  much  less  response  to  wind  than  the  atmometer,  but  with 
the  sudden  advent  of  a  high  wind  it  often  showed  greater  response. 

12.  Wind  carries  dust  to  the  leaves,  coating  them  and  often 
wedging  particles  into  the  open  stomatal  sUts,  so  that  they  remain 
permanently  open. 

13.  Water-content  is  the  chief  of  the  factors  that  determine  the 
rate  at  which  the  leaves  can  be  supplied  with  water.  When  the 
jBoil  is  dry  the  rate  of  supply  is  slow,  causing  turgor  to  be  lost  early 
jn  the  day  and  the  stomata  to  close. 


76         EFFECT  OF  PHYSICAL  FACTORS  AND  PLANT  CONDITIONS. 

14.  Water-logging  the  soil  causes  closure  of  the  stomata  and  sub- 
sequent wilting  by  inhibiting  the  functioning  of  the  roots. 

15.  The  maximum  of  leaf  turgor  in  most  cases  is  reached  about 
midnight.  After  stomatal  opening  at  daybreak,  or  shortly  after- 
ward, it  begins  to  decrease,  the  rate  being  dependent  upon  transpi- 
ration on  the  one  hand  and  water-supply  on  the  other.  Part  of 
the  water  present  in  the  leaf  at  the  start  is  a  working  margin  and 
the  stomata  do  not  close  until  this  is  gone.  If  the  roots  can  keep 
up  a  sufficient  rate  of  supply  this  working  margin  does  not  wholly 
disappear  during  the  day  and  the  stomatal  movement  is  of  the  nor- 
mal light-induced  type.  If  the  margin  is  lost,  however,  the  stomata 
close  until  it  is  recovered,  at  least  in  part. 

16.  The  nightly  maximum  of  leaf  turgor  increases  after  every 
rain  or  irrigation  and  thereafter  decreases  until  the  next  rain.  Such 
fluctuation  between  rains  is  often  very  great.  In  lesser  degree,  the 
critical  leaf-water  or  percentage  at  which  the  working  margin  disap- 
pears also  fluctuates  between  rains  or  irrigations. 

17.  The  degree  of  succulence  of  a  leaf  determines  to  a  large  extent 
the  amount  of  water  present  as  a  working  margin,  and  therefore 
the  readiness  with  which  the  stomata  close  when  transpiration  is  high. 

18.  The  growth  habit  of  a  plant  often  affects  stomatal  movement 
by  reserves  of  water  to  increase  the  rate  of  supply  during  periods  when 
loss  is  greatest  and  by  roots  which  penetrate  to  permanently  moist  soil. 

19.  The  age  or  degree  of  maturity  of  the  plant  affects  its  stomatal 
movement,  and  the  age  of  each  leaf  influences  the  readiness  with 
which  the  stomata  on  it  function. 

20.  The  number  and  size  of  the  stomata  on  any  given  area  of 
leaf  are  influenced  by  the  conditions  under  which  they  were  formed. 
A  leaf  developed  in  the  shade  has  fewer  and  larger  stomata  per 
unit  area  than  one  produced  in  sunlight. 

21.  The  removal  of  the  hairs  from  the  leaf  of  a  plant  with  very 
hairy  leaves  caused  the  stomata  to  open  somewhat  earlier  upon  the 
appearance  of  light  and  close  very  much  sooner,  showing  that  the 
water-loss  from  such  a  leaf  was  probably  much  higher  than  from 
the  normal  leaves. 

22.  Plants  of  desert  regions  have  adapted  themselves  to  their 
surroundings  in  two  ways,  so  far  as  stomatal  movement  is  con- 
cerned. Some  produce  a  crop  of  leaves  when  opportunity  per- 
mits, which  wither  and  disappear  when  the  water-supply  becomes 
critical.  Such  leaves  are  mesophytic  in  type  as  a  rule,  and  show 
no  specialization  for  desert  conditions.  Other  plants  have  per- 
sistent leaves,  or  stems  adapted  for  photosynthetic  work,  which  show 
considerable  adaptation  to  desert  conditions,  hairs,  wax,  auiiken 
stomata,  and  other  means  being  used  to  reduce  water-loss.  Such 
plants  often  normally  show  a  reversal  of  the  usual  beEavior  of  the 
stomata,  opening  occurring  at  night  and  closure  during  the  day^ 
as  in  many  mesophytes  when  exposed  to  similar  arid  conditions. 


III.  THE  EFFECT  OF  STOMATAL  MOVEMENT  UPON 
TRANSPIRATION. 

Indirect  evidence  that  stomatal  movement  affects  the  water-loss 
of  plants  had  gradually  accumulated  during  the  course  of  the  experi- 
ments. This  evidence,  however,  was  not  conclusive  and  in  some 
instances  was  capable  of  several  interpretations.  For  this  reason  a 
number  of  experiments  were  made  to  discover  the  relationship 
between  stomatal  movement  and  transpiration,  and  to  find  some 
reason  for  divergence  of  views  concerning  the  effectiveness  of  the 
stomatal  regulation  of  water-loss.  It  seemed  inconceivable  that 
complete  closure  of  the  stomata  should  fail  to  reduce  the  rate  of 
transpiration  to  a  considerable  extent,  as  Buscalioni  and  Polacci  (1902) 
have  confirmed  the  earlier  results  as  to  the  relative  insignificance  of 
cuticular  transpiration  by  showing  that  a  collodion  film  placed  on 
a  leaf  clouded  quickly  just  above  the  stomata  only.  In  the  course  of 
several  series,  plants  were  often  found  to  wilt  visibly  during  the  early 
forenoon,  and  the  stomata  to  close,  as  a  consequence  of  which  they 
recovered  turgor  by  afternoon,  when  the  stomata  again  opened.  The 
recovery  of  turgor  during  the  part  of  the  day  when  evaporation  was 
greatest  must  be  ascribed  to  closure  of  the  stomata.  The  commonly 
accepted  view  that  stomata  are  not  regulatory  was  not  in  accord 
with  these  facts,  but  further  investigation  was  imperative  to  fully 
explain  the  divergence  of  views. 

As  a  result  of  the  researches  of  Mohl  (1856),  Merget  (1873),  Schwen- 
dener  (1881),  Leitgeb  (1888),  Stahl  (1894),  Kohl  (1895),  Darwin 
(1898),  and  many  others,  the  mechanism  of  the  stomata  was  demon-  c^ 
strated,  and  they  were  shown  to  be  the  outlets  for  water-vapor 
and  hence  of  the  greatest  importance  in  transpiration.  In  conse- 
quence, the  view  came  to  be  held  that  the  stomata  by  their  move- 
ments completely  controlled  the  water-loss  from  the  plant,  except 
for  the  inconsiderable  amount  evaporated  from  the  cuticle.  It  was 
apparently  believed  that  the  air-spaces  of  the  leaf  contained  a 
water-saturated  gas  with  but  Httle  CO2  and  the  vapor  diffused  out 
through  the  stomata  as  the  CO2  passed  in.  Under  such  conditions 
the  evaporating  power  of  the  air,  especially  when  moving,  would 
have  little  effect  upon  transpiration  and  the  stomata  would  be  wholly 
regulatory.  Brown  and  Escombe  (1900),  however,  showed  that  the 
theoretical  diffusion  of  water-vapor  from  a  leaf  of  Helianthus 
annuus  was  about  six  times  as  great  as  that  actually  lost  by  the  plant. 
The  full  significance  of  this  discovery  was  overlooked,  however,  in 
view  of  the  expressed  doubt  that  the  stomata  were  of  sufficient  size 
and  number  to  permit  such  large  amounts  of  CO2  and  water-vapor 

77 


78        EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 

to  pass  through  by  diffusion  alone.  The  authors  showed  that  no 
theory  of  streaming  currents  into  the  leaf  or  from  it  was  needed. 
Lloyd  (1008)  apparently  was  the  first  to  point  out  that  since  this 
rate  of  diffusion  is  much  greater  than  the  rate  of  transpiration,  the 
gas  inside  the  leaf  can  not  be  fully  saturated: 

"The  actual  rate  of  diffusion  through  stomata  will  depend  upon  the  length 
of  the  tube,  gradation  of  density  of  the  water-vapor  between  the  surface  of 
the  cells  of  the  chlorenchyma  and  the  outer  air,  modified  by  air-currents. 
It  seems  clear  from  Brown  and  Escombe's  experiment  that  there  exists  such 
a  gradation  of  density,  from  which  it  follows  that  under  conditions  of  rapid 
transpiration  the  vapor-pressure  within  the  leaf  cavities  is  less  than  when 
a  low  rate  occurs,  assuming  the  evaporation  capacity  of  the  cells  to  be  con- 
stant. As  in  the  case  of  CO2,  the  pressure  of  the  water- vapor  within  the 
leaf,  though  much  greater,  must  vary  with  the  size  of  the  stomatal  pores, 
but  here  the  relative  humidity  without  is  a  very  variable  factor  and  will, 
therefore,  modify  the  rate  of  transpiration  independently  of  the  pores." 

In  this  Lloyd  summed  up  the  entire  problem  of  the  water -loss 
from  a  plant-leaf. 

The  problem  of  stomatal  regulation  is  therefore  comphcated  by 
several  conditions,  the  chief  of  which  is  the  changing  vapor-pressure 
of  the  air-spaces  under  different  conditions  of  stomatal  opening, 
relative  humidity,  wind,  radiant  energy,  and  air-temperature.  Tre- 
lease  and  Livingston  (1916)  believe  that  changes  in  the  chloren- 
chyma reduce  transpiration  before  stomatal  closure  occurs.  As  this 
behef  is  based  upon  the  "relative  transpiration"  calculated  from  the 
evaporation-rate  of  the  porous-cup  atmometer,  it  has  not  been 
demonstrated  conclusively.  Nevertheless,  it  is  probable  that  this 
condition  also  enters  into  the  problem  and  adds  its  complications  to 
it.  The  problem  of  stomatal  regulation  of  transpiration,  therefore, 
can  not  be  confined  to  the  thesis  that  small  changes  of  apertures 
produce  like  changes  in  the  absolute  rate  of  transpiration.  Stomata 
must  be  considered  definitely  regulatory,  if  changes  in  their  openings 
produce  similar  changes  in  the  effectiveness  upon  the  leaf  of  the 
evaporating  power  of  the  air. 

The  problem  was  attacked  from  another  angle  by  Muenscher 
(1915),  who  compared  the  water-loss  from  9  species  of  plants  with 
the  size  and  number  of  their  stomata.  While  the  investigation 
apparently  showed  no  relationship  between  transpiration  and  stoma- 
tal opening,  the  results  are  vitiated  by  the  failure  to  take  into  account 
the  length  of  time  the  stomata  of  each  plant  were  open.  The  follow- 
ing table  is  taken  from  his  paper. 

Table  3.  Relation  between  aynount  of  transpiration  and  stomatal  aperture  (from  Muenscher). 


Linear  units  of  etomatal  pore  in  microns  per  sq.  mm . .  . 
Amount  of  transpiration  per  sq.  dm.  per  hour  in  milligrams. 
Area  of  stomatal  apertures  per  sq.  mm.  leaf  surface 


Zea  mays. 


1,530 

80 

3.864 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION.        79 

The  ratio  of  2,056  linear  units  in  sunflower  to  1,530  linear  units  in 
corn  is  approximately  4  : 3,  while  the  transpiration  per  unit  surface 
of  sunflower  is  nearly  twice  that  of  corn.  Only  when  the  assumption 
is  made  that  the  stomata  of  both  species  of  plants  are  open  all  the 
time,  or  are  open  equally  for  the  same  period,  can  this  be  taken  as 
evidence  that  the  diffusion  capacity  of  the  stomata  does  not  regulate 
transpiration.  Muenscher's  plants  were  under  far  different  condi- 
tions of  environment  from  those  used  in  this  investigation,  but  a 
recalculation  of  results  based  upon  the  present  findings  is  of  interest. 
A  series  containing  both  Zea  mays  and  Helianthus  annuus  growing 
under  the  same  conditions  showed  the  stomata  of  corn  open  approx- 
imately 8  hours  and  those  of  sunflower  12  hours.  If  this  was  true 
for  Muenscher's  plants,  disregarding  cuticular  water-loss,  the  amount 
transpired  during  a  24-hour  period  must  have  been  lost  largely 
during  12  hours  in  Helianthus  and  8  hours  in  Zea  mays.  The  hourly 
water-loss  for  Helianthus  would  then  be  312  mg.  and  for  Zea  mays 
240  mg.  per  hour  per  square  decimeter.  The  ratio  of  linear  units  for 
sunflower  and  corn  is  approximately  20  :  15  and  the  ratio  of  hourly 
transpiration  recalculated  in  this  manner  312  :  240,  both  of  which 
reduce  approximately  to  4  :  3.  Correction  for  cuticular  water-loss 
would  undoubtedly  make  the  agreement  very  close.  However,  this 
explanation  is  only  of  theoretical  interest;  the  preceding  sections 
have  shown  the  futility  of  attempting  to  estimate  the  stomatal 
behavior  of  a  plant  in  an  unknown  environment.  It  does  show,  how- 
ever, why  Muenscher  can  not  be  considered  to  have  produced  valid 
evidence  to  the  effect  that  "the  amount  of  transpiration  is  not  gov- 
erned entirely  by  stomatal  regulation." 

In  spite  of  the  care  with  which  Lloyd  (1908)  carried  out  his  ex- 
periments, and  the  clearness  of  his  analysis  of  the  problem,  he  can 
not  be  said  to  have  proved  the  case  against  stomatal  regulation  with 
any  greater  success.  He  realized  that  the  use  of  potometers  to  meas- 
ure transpiration  was  the  weak  part  of  his  investigation,  but  did 
not  check  them  sufficiently  to  ascertain  where  this  weakness  lay. 
His  discovery  that  the  amount  of  water  lost  from  the  stem  and  leaves 
was  not  the  same  as  that  absorbed  possibly  caused  him  to  overlook 
the  very  important  fact  that  the  stomatal  movement  in  the  leaves  of 
a  cut  stem  is  not  at  all  hke  that  of  the  leaves  of  a  field  plant  or  even 
a  potted  plant.  Hence  Lloyd  assumed  inadvertently  the  very  thing 
he  attempted  to  prove,  namely,  that  stomatal  movement  had  no 
effect  upon  transpiration. 

To  gain  a  clearer  idea  of  the  reliability  of  cut  stems  as  a  meas- 
ure of  water-loss  from  rooted  plants,  an  alfalfa  series  was  made  on 
August  8,  1916.  A  battery  of  potometers  was  set  up  and  a  pre- 
liminary experiment  carried  out  on  August  5  to  determine  whether 
alfalfa  stems  treated  in  this  manner  would  check  with  one  another 
in  regard  to  their  transpiration  (plate  11).    The  burette  in  each  case 


80        EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 

was  connected  through  a  glass  tee  to  the  plant  on  one  hand  and  a 
flask  used  as  a  reservoir  on  the  other.  A  pinch-cock  permitted  the 
refilling  of  the  burette  from  the  reservoir  after  each  reading;  this 
constantly  maintained  the  water-pressure  upon  the  cut  end  of  the 
stem  at  nearly  the  same  level.  Other  precautions  were  observed;  the 
entire  apparatus  was  sterilized  with  4  per  cent  formalin,  and  the 
water  used  was  recently  distilled  and  boiled  just  before  using.  The 
stem  of  the  plant  used  was  sterilized  at  the  point  desired,  and  cut 
under  water  with  a  sterile  knife.  The  cut  end  of  the  stem  was  pro- 
tected by  a  loose  plug  of  absorbent  cotton.  These  potometers  were 
run  28  hours  and  read  at  half -hour  and  hour  intervals  during  the 
two  days  and  at  the  end  of  a  10-hour  night  interval.  Since  the  read- 
ings ran  in  parallel  series,  the  test  was  considered  satisfactory.  The 
stems  used  were  then  discarded  and  the  apparatus  resterilized. 

At  4  p.  m.  on  August  8,  the  potometers  were  started  with  fresh 
stems,  which  were  gathered  with  all  the  precautions  observed  when 
making  the  trial.  Readings  were  made  at  half-hour  intervals  until 
7  p.  m.  to  insure  that  the  potometers  were  working  properly.  As  it 
was  found  to  be  impossible  to  check  the  transpiration  from  the  cut 
stems  against  that  of  field  plants,  or  even  of  potted  plants,  it  was 
decided  to  compare  the  stomatal  movement  of  such  cut  stems  with 
the  stomatal  movement  of  field  plants.  For  this  purpose,  30  branches 
of  alfalfa  were  cut  under  water  with  the  usual  precautions.  These 
were  placed  in  flasks  of  the  same  kind  of  water  used  in  the  burettes. 
In  order  to  find  whether  the  head  of  water  against  the  cut  ends  of  the 
stems  in  the  potometers  had  some  effect  upon  the  stomatal  movement 
not  found  in  those  in  the  flasks,  several  stems  were  treated  in  the 
same  manner  as  those  in  the  potometers,  using  plain  glass  tubing 
instead  of  burettes.  A  sufficient  number  of  these  were  set  up  so 
that  strips  could  be  taken  at  4-hour  intervals  during  the  series.  As 
may  be  expected,  the  increased  water-pressure  on  the  cut  ends  did 
not  have  sufficient  effect  to  make  a  discernible  difference  in  the 
behavior  of  the  stomata.  The  leaves  of  one  stem  were  stripped  each 
hour  and  the  stem  immediately  discarded  to  prevent  its  being  used 
again. 

A  sHght  and  misty  rain  occurred  between  10  and  lip.  m.,  but  by 
midnight  the  weather  had  cleared.  At  1  a.  m.  the  wind  had  risen 
and  was  drying  the  vegetation.  By  2  a.  m.,  when  the  wind  died 
down,  all  effects  of  the  rain  had  disappeared,  and  the  night  remained 
clear  and  still  from  that  time.  The  following  day  was  warm,  prac- 
tically cloudless,  and  more  humid  than  ordinarily,  since  the  relative 
humidity  did  not  fall  below  28  per  cent.  Although  it  was  a  sunny 
day,  haze  prevented  the  light  from  reaching  more  than  75  per  cent 
of  the  maximum  intensity  for  the  region.  As  usual,  the  lowest 
temperature,  55°  F.,  was  recorded  at  5  a.  m.,  just  before  dawn.  The 
highest  was  78°  F.,  occurring  at  3  p.  m.  the  same  day  (fig.  37). 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION.       81 

The  difference  in  the  stomatal  movement  of  the  cut  stems  and  the 
watered  field  plants  was  striking.  The  watered  plants  showed  15 
and  20  per  cent  opening  from  10  p.  m.  until  1  a.  m.,  and  closure  from 
then  to  dawn.    This  was  largely  the  result  of  the  slight  rain  which 


' 

/ 

\ 

,0 

_ 

/ 

^ 

^ 

...• 



_. 

n' 

7" 

^^ 

N. 



-., 



. 



-=.- 

'' 

B 

V 

wx 

^ 

^f 

/ 

-■ 

-' 

■••• 

J\ 

\ 

/ 



f 

\ 

/ 

/ 

/ 

^ 

-, 

c 

\/ 

/ 

"n 

— 

-" 

"" 

/ 

\ 

n 

J 

_J 

_ 

IT.    I       2      3 


5      6      7      8      3 


II  NOON  1       23456789     lO 


Fig.  37. — Series  17,  weather  data  for  Augu.st  8-9,  1916;  .sunlight  (A), 
temperature  (B),  humidity  (C). 


/             \ 

lX-            -^ 

-4      1-                      t 

73    I             A 

^14              ^ 

2\       ^    ^    A                  X 

Jl^  7  4(      3               3 

^^      S/    I   C   ^ 

^  \.^    s  x  s^       X 

^-A-^^7\2      1   /""^^--^S   ^^Z 

2      3      -4.      5 


7      8      9      10 


34.56789      10 


Fig.  38. — Series  17,  showing  average  movement  of  alfalfa  stomata  in  watered 
(A)  and  unwatered  (B)  field  plants,  and  in  cut  stems  (C). 


S       6       7      8       9      10     "  NOON 


5      6      7      8      9      10 


Fig.  39. — Series  17,  showing  stomatal  movement  (A),  and  transpiration  (B) 
in  cut  stems  of  alfalfa;  humidity  (C). 

occurred  after  10  p.  m.  The  cut  stems  had  closed  stomata  at  10  p.  m., 
but  they  opened  slowly  to  10  per  cent  at  1  a.  m.  and  then  began 
closing.     At  3  a.  m.  this  was  complete.     The  next  hour,  however, 


82        EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 

they  opened  10  per  cent  and  closed  again  at  5  a.  m.  The  sun  appeared 
through  the  clouds  over  the  mountains  at  5*"  45°*  a.  m.,  but  it  began 
to  be  hght  shortly  after  5  a.  m.  In  consequence,  the  stomata  of  the 
field  plants  opened  10  per  cent  at  6  a.  m.,  40  per  cent  at  7,  80  per 
cent  at  8,  and  were  wide  open  by  9  a.  m.  The  stomata  of  the  cut 
stems  acted  in  practically  the  same  manner,  except  that  opening 
was  somewhat  slower  at  the  start.  Maximum  opening  lasted  until 
2  p.  m.  in  the  field  plants.  The  stomata  of  the  cut  stems,  however, 
closed  immediately  to  25  per  cent  at  10  a.  m.,  10  per  cent  at  11  a.  m. 
and  noon,  and  then  slowly  to  5  per  cent  at  2  p.  m.  They  remained  in 
this  condition  until  3  p.  m.,  and  then  closed  very  slowly  and  com- 
pletely by  5  p.  m.,  remaining  closed  to  the  end  of  the  series.  The 
watered  field  plants  started  to  close  after  2  p.  m.  This  continued 
uniformly  throughout  the  afternoon,  and  was  completed  at  6  p.  m. 
(fig.  38). 

The  stomatal  movement, of  the  unwatered  field  plants  showed  even 
greater  divergence  from  that  of  the  cut  stems.  The  considerable- 
night  opening  found  in  these  plants  did  not  occur  in  the  cut  stems, 
and  the  maximum  opening  at  7  a.  m.  was  2  hours  earlier  than  that 
found  in  either  the  heavily  watered  field  plants  or  the  cut  stems. 
Complete  closure  occurred  at  9  a.  m.,  when  the  stomata  of  the  cut 
stems  and  watered  plants  had  reached  maximum  opening  (fig.  38). 
It  must  be  concluded,  therefore,  that  the  water-loss  from  alfalfa 
potometers  can  not  be  used  as  a  measure  of  the  water-loss  from 
naturally  growing  plants,  nor  can  stomatal  regulation  be  judged 
ineffective  by  reason  of  such  comparison. 

The  transpiration  of  the  cut  stems  as  measured  in  the  potometers 
is  distinctly  controlled  by  stomatal  movement.  For  several  reasons, 
the  correlation  between  the  rate  of  water-loss  and  the  degree  of 
stomatal  opening  is  among  the  best  found  in  these  experiments.  The 
compUcations  entering  into  the  problem  by  sudden  changes  of  wind 
velocity,  wide  fluctuations  of  relative  humidity  within  brief  inter- 
vals, and  sudden  changes  of  sunlight  caused  by  passing  clouds  did 
not  occur.  In  addition,  the  difference  between  the  maximum  and 
minimum  temperatures  was  not  nearly  as  great  as  usual,  changes  were 
slow  and  gradual,  and  the  temperature  remarkably  constant  during 
the  greater  part  of  the  day.  The  stomatal  opening  described  in 
connection  with  transpiration  in  this  section  is  the  average  for  both 
surfaces  of  the  leaf,  calculated  for  the  number  of  stomata  per  unit 
area  on  each  surface. 

Between  10  and  11  p.  m.  the  average  water-loss  from  the  alfalfa 
potometers  or,  more  accurately,  the  water  absorbed,  was  7.0  mg. 
per  minute  per  square  decimeter  of  leaf-surface,  which  is  the  upper 
and  lower  surface  of  0.5  square  decimeter  of  leaf  area.  During  this 
time  the  stomata  opened  to  4  per  cent,  most  of  the  opening  occurring 
in  the  upper  surface.     During  the  next  hour  the  water-loss  was 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION.        83 

6.7  mg.  per  minute,  while  the  stomata  remained  at  5  per  cent  opening. 
From  midnight  to  1  a.  m.  the  average  loss  was  8.7  mg.  per  minute, 
corresponding  to  an  increase  to  10  per  cent  opening  of  the  stomata. 
Between  1  and  2  a.  m.  the  rate  of  loss  decreased  to  7.5  mg.  per 
minute,  while  the  stomata  closed  to  5  per  cent.  A  fall  in  the  rate  to 
4.5  mg.  during  the  next  hour  occurred  as  the  stomata  closed.  As  the 
stomata  opened  to  10  per  cent  at  4  a.  m.  the  average  rate  of  trans- 
piration was  4.2  mg.,  but  as  a  result  of  this  opening  it  rose  to  6.7 
mg.  between  4  and  5  a.  m.  At  5  a.  m.  the  stomata  closed  again  and 
the  rate  of  loss  dropped  to  1.5  mg.  This  very  low  rate  was  also  due 
to  the  rise  of  relative  humidity  to  80  and  90  per  cent.    Between  6  and 

7  a.  m.  the  stomata  opened  from  5  to  35  per  cent  and  the  rate  of 
transpiration  rose  to  13.5  mg.  The  following  hour  opening  increased 
from  35  to  80  per  cent,  relative  humidity  fell  below  70  per  cent,  and, 
in  consequence,  the  rate  increased  to  47.5  mg.  per  minute.    Between 

8  and  9  a.  m.  the  stomata  opened  from  80  per  cent  to  maximum,  and 
the  rate  of  loss  to  100  mg.  per  minute.  The  next  hour  the  stomata 
closed  to  25  per  cent  and  transpiration  fell  to  37.0  mg.  This  was  in 
spite  of  the  fact  that  relative  humidity  had  fallen  to  46  per  cent  and 
sunlight  had  reached  the  average  maximum  for  the  day.  Further 
closure  to  10  per  cent  at  11  a.  m.  was  accompanied  by  a  fall  to  26.7 
mg.  per  minute.  From  this  time  slow  closure  of  the  stomata,  com- 
pleted at  5  p.  m.,  was  accompanied  by  a  similar  fall  in  the  transpira- 
tion-rate. The  stomata  were  closed  from  5  to  10  p.  m.,  at  which 
time  the  series  ended.  The  rate  of  water-loss  remained  rather  con- 
stant from  5  to  7  p.  m.,  since  the  relative  humidity  was  also  as  con- 
stant, but  after  7  p.  m.  the  humidity  rose  rapidly  and  the  trans- 
piration-rate again  fell.  Between  9  and  10  p.  m.,  when  the  humidity 
again  became  fairly  stable,  changing  only  from  74  to  76  per  cent, 
transpiration  decreased  only  from  4.7  to  4.5  mg.  per  minute. 

The  correlation  in  this  experiment  between  the  stomatal  movement 
and  the  rate  of  transpiration  of  cut  stems  is  remarkably  clear. 
Where  there  is  deviation  in  the  rate  of  water-loss  from  the  changes 
of  stomatal  opening  this  is  clearly  due  to  fluctuations  of  relative 
humidity.  When  the  fluctuations  are  rapid  and  irregular  they 
introduce  complications  which  are  hard  to  measure.  But  in  this 
experiment  the  humidity  changed  slowly  and  rather  uniformly,  and 
was  nearly  constant  for  long  periods.  Because  this  did  not  occur 
in  the  other  experiments,  and  because  of  other  reasons,  the  later 
series  failed  to  show  as  clear  a  relation  between  stomatal  movement 
and  transpiration.  The  stomatal  movement  of  the  field  plants, 
watered  and  unwatered,  did  not  in  any  manner  resemble  that  of  the 
cut  stems,  and  the  transpiration  from  the  potometers  in  turn  showed 
no  relationship  to  the  changes  in  stomatal  opening  of  these  plants. 
For  both  these  reasons,  in  the  case  of  alfalfa  at  least,  potometers 
can  not  be  used  to  represent  the  transpiration  of  rooted  plants. 


84        EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 

Another  series  was  made  with  the  Russet  Burbank  potato  instead 
of  alfalfa,  in  order  to  determine  the  reliability  of  potometers  in  the 
case  of  this  species.  The  potometers  were  fitted  up  with  the  same 
care  observed  in  the  earher  series  in  which  alfalfa  was  used.  In 
this  case  the  stomatal  movement  of  the  cut  stems  was  compared 
with  that  of  potted  plants,  as  well  as  with  watered  and  unw^atered 
field  plants,  to  discover  if  the  cut  steps  would  show  movement  more 
nearly  resembling  that  found  in  the  potted  plants  than  the  field 
plants.  As  no  scales  of  sufficient  capacity  were  available  to  weigh  the 
very  large  pots  closely  enough  to  measure  their  water-loss,  only  the 
transpiration  from  the  cut  stems  was  measured.  The  experiment 
(series  20)  was  started  at  noon  on  August  25  and  ended  at  1  p.  m. 
on  August  26,  1916.  The  potometers  were  set  up  and  the  first  readings 
made  at  10  a.  m.  on  the  25th,  in  order  to  find  whether  each  was 
working  properly,  before  the  series  was  begim.  As  the  readings 
ran  in  a  parallel  series,  they  were  considered  as  functioning  properly, 
and  each  potometer  read  to  the  end  of  the  experiment  the  next 
day.  As  in  the  case  of  the  alfalfa  plants,  the  leaves  were  then  clipped 
off  and  printed  on  Solio  paper,  and  the  total  area  determined  for 
each  stem  separately  by  means  of  a  planimeter.  An  error  enters 
in  at  this  point,  because  the  stems  also  have  functional  stomata, 
but  their  number  is  not  great  and  they  can  not  have  much  more 
influence  than  cuticular  water-loss.  As  the  area  of  stem-surface 
in  proportion  to  leaf-area  was  nearly  the  same  in  each  potometer, 
the  effect  of  the  stem-surface  could  not  be  determined.  Hence,  it 
would  be  more  accurate  to  state  that  the  water-loss  given  was  that 
from  1  sq.  dm.  of  leaf -surface  and  1.62  sq.  cm.  stem-surface.  As, 
however,  no  attempt  is  made  to  compare  the  transpiration  of  several 
plants,  the  unit  of  area  from  which  the  water-loss  occurs  is  immaterial, 
providing  it  is  not  changed  during  the  experiment.  The  real  problem 
is  whether  the  changes  in  the  rate  of  transpiration  from  some  given 
area,  regardless  of  its  size,  shows  any  relationship  to  changes  in  the 
stomatal  openings. 

The  temperature,  relative  humidity,  and  sunlight  fluctuated  a 
great  deal  during  the  experiment.  The  two  days  were  rather  hot, 
the  highest  temperatures  being  87°  F.  at  3  p.  m.  on  August  25  and 
88°  F.  at  1  p.  m.  on  the  26th.  As  usual,  the  lowest  temperature, 
57.5°  F.,  was  recorded  at  5  a.  m.,  just  before  dawn.  The  humidity 
dropped  to  19  per  cent  at  4  and  5  p.  m.  on  the  25th,  about  one-third 
lower  than  that  recorded  during  the  alfalfa  series.  Inconstant  winds 
caused  irregular  decreases  of  humidity  during  the  night,  and  the 
maximum  of  71  per  cent  occurred  several  times.  During  the  greater 
part  of  the  two  days  there  was  a  gentle  breeze,  with  occasional  gusts 
of  strong  winds,  which  increased  transpiration  during  short  intervals. 
There  were  occasional  periods  of  calm,  which  also  affected  water- 
loss.    Sunlight  was  very  variable  because  of  changing  haziness.    On 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 


85 


August  25  there  was  65  per  cent  light  at  noon,  which  decreased  to 
53  per  cent  at  2  p.  m.  At  3  p.  m.  the  intensity  had  increased  to 
60  per  cent  and  then  decreased  uniformly  until  sundown  at  7  p.  m. 
The  sun  rose  above  the  mountains  shortly  after  6  a.  m.  August  26, 
but  clouds  prevented  the  light  from  reaching  more  than  4  per  cent 
at  7  a.  m.  At  7^  30™  it  was  9  per  cent,  28  per  cent  at  8,  and  43  per 
cent  at  8^  25™  a.  m.  At  9  it  reached  55  per  cent,  but  deepening 
haze  prevented  a  rapid  increase  in  intensity  for  a  time.  At  noon  the 
haze  began  to  clear,  and  at  1  p.  m.  the  light  reached  88  per  cent,  the 
maximum  during  the  period  of  the  experiment  (fig.  40). 

All  the  plants,  except  the  heavily  watered  field  plants,  suffered 
from  excessive  water-loss.  The  cut  stems  had  open  stomata  from 
10  a.  m,  until  1  p.  m.,  when  the  lower  stomata  started  to  close, 
reaching  15  per  cent  at  2  p.  m.,  while  no  change  occurred  in  the  upper. 
At  3  p.  m.  the  upper  closed  to  one-half  of  their  maximum,  while  the 
lower  opened  to  25  per  cent.  The  upper  closed  to  40  per  cent  and  the 
lower  to  20  per  cent  at  4  p.  m.  At  5  p.  m.  the  lower  closed  to  10 
per  cent  and  the  upper  to  20  per  cent.    At  6  p.  m.  the  lower  opened 


90 

"s^                             .--  J 

^                                                  ^-C"^/'^ •'               ^>                  -y-^                         ^ 

I    ■\7\^        /        -       -= ^-^-^^ 

-          S  ^                              A^ 

^i.          ^I                              t  '^ 

Z—.^     -S'                           ^       "■- 

""t                        2 

\                      ^^ 

7       8      9      10      II  NOON 


FiQ.  40. — Series  20,  weather  data  for  August  25-26,  1916;  sunlight  (A),  temper- 
ature (B),  humidity  (C). 

to  20  per  cent,  while  the  upper  remained  in  the  same  condition.  At 
7  p.  m.  the  upper  opened  to  25  per  cent  and  the  lower  closed  to  10 
per  cent.  At  8  p.  m.  the  lower  stomata  closed,  while  the  upper  were 
20  per  cent  open.  At  9  p.  m.  the  upper  closed  to  15  per  cent  and  at 
10  p.  m.  closed  completely.  At  this  time,  however,  the  lower  opened 
3  per  cent,  and  5  per  cent  at  11  p.  m.  By  midnight  all  closed,  except 
occasional  stomata  in  both  surfaces,  producing  1  or  2  per  cent  opening 
throughout  the  rest  of  the  night.  At  6  a.  m.  the  upper  stomata  opened 
10  per  cent,  and  20  per  cent  at  7  a.  m.  At  9  a.  m.  the  lower  opened 
5  per  cent,  while  the  upper  remained  in  the  same  condition.  At  10 
a.  m.  the  lower  closed  to  2  per  cent,  in  which  condition  they  remained 
until  noon.  The  upper  closed  to  15  per  cent  at  this  time,  then  to 
5  per  cent  at  11  a.  m.  and  remained  in  this  condition  until  noon. 
At  1  p.  m.  the  stomata  of  both  surfaces  closed  completely^  (fig.  41). 


86        EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 

The  stomata  of  the  potted  plants  were  wide  open  at  the  start  and 
remained  open  an  hour.  The  lower  stomata  then  closed  completely 
by  3  p.  m.,  while  the  upper  stomata  closed  to  20  per  cent  at  5  p.  m. 
No  further  change  took  place  in  either  surface  until  after  7  p.  m. 
At  8  p.  m.  the  upper  started  to  reopen  slightly,  reaching  30  per  cent 
at  10  p.  m.  At  midnight  further  opening  again  took  place,  the  upper 
showing  50  per  cent  opening.  At  1  a.  m.  the  lower  showed  20  per  cent 
opening,  and  the  upper  80  per  cent;  at  2  a.  m.  the  lower  were  half 
open  and  the  upper  at  maximum.  At  3  a.  m.  the  stomata  of  both 
surfaces  were  wide  open,  and  so  remained  to  the  end  of  the  series. 


\ 

r- 

: 

, 

00 

\ 

: 

/ 

/ 

,3 

\  \ 

/ 

: 

/ 

' 

\ 

A,- 

B/ 

\ 

/ 

/ 

Y 

< 

^ 

>^- 

.... 

/ 

.: 

A 

\ 

/ 

\ 

\ 

^ 

^- 

-- 

^'' 

/ 

^s 

\, 

^ 

■^ 

::n 

■^ 

_ 

^ 

z 

^ 

-::^ 

^ 

Fig.  41. — Series  20,  showing  movement  in  upper  (A)  and  lower  (B)  stomata  of 
potted  potato  plants,  and  in  upper  (C)  and  lower  (D)  stomata  of 
cut  potato  stems. 

The  stomata  of  the  watered  field  plants  were  wide  open  until 
4  p.  m.,  when  the  lower  began  to  close.  At  5  p.  m.  the  upper  also 
showed  closure  to  80  per  cent,  while  the  lower  were  but  40  per  cent. 
At  6  p.  m.  the  lower  were  closed  and  the  upper  were  50  per  cent 
open.  At  7  p.  m.  the  upper  closed  to  30  per  cent,  and  at  8  p.  m.  closed 
completely.  At  midnight  the  upper  showed  50  per  cent  opening, 
and  complete  opening  the  following  hour.  The  lower  were  but  30 
per  cent  open  at  midnight,  60  per  cent  at  1  a.  m.  and  wide  open 
at  2  a.  m.  They  remained  at  maximum  to  the  end  of  the  series. 
The  unwatered  field  plants  had  closed  stomata  when  the  series 
began.  At  2  p.  m.  the  lower  opened  10  per  cent,  closed  to  5  per  cent 
at  3,  and  opened  to  15  per  cent  at  4  p.  m.  At  this  time  the  upper 
opened  to  20  per  cent,  and  to  30  per  cent  at  5  p.  m.,  while  the  lower 
closed  to  5  per  cent  at  this  time.  At  6  p.  m.  the  lower  closed  com- 
pletely and  the  upper  to  15  per  cent.  At  7  p.  m.  both  were  closed, 
but  at  8  the  upper  reopened  5  per  cent.  At  9  p.  m.  the  lower  opened 
10  per  cent,  while  the  upper  reached  25  per  cent.  At  10  p.  m.  both 
surfaces  showed  20  per  cent  opening,  and  closure  at  11  p.  m.  At 
midnight  the  stomata  of  the  upper  surface  opened  25  per  cent,  those 
of  the  lower  10  per  cent,  and  at  1  a.  m.  the  upper  reached  60  per  cent 
and  the  lower  30  per  cent.  At  2  a.  m.  the  upper  were  80  per  cent 
open,  but  closed  to  70  per  cent  the  following  hour.     In  the  mean- 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION.        87 

time,  the  lower  opened  to  50  per  cent  at  2  a.  m.  and  70  per  cent  at  3. 
At  4  a.  m.  both  surfaces  showed  50  per  cent  opening,  and  remained 
in  this  condition  for  an  hour.  At  6  a.  m.  the  lower  were  but  40  per 
cent  open,  while  the  upper  opened  to  90  per  cent.  At  7  a.  m.  the 
lower  opened  to  70  per  cent,  while  the  upper  were  wide  open.  At 
8  a.  m.  the  stomata  of  both  surfaces  were  wide  open  and  so  remained 
until  10  a.  m.,  when  the  lower  closed  to  70  per  cent.  At  11  a.  m.  the 
upper  closed  to  90  per  cent  and  the  lower  to  30  per  cent.  At  noon  all 
the  stomata  were  closed  and  remained  closed  to  the  end  of  the  series 
(fig.  42). 


90 -V\ 

P        ^  ■■    ■  "^    " 

80                             '      ^ 

U         4    ■■'■ 

70                                 X-\-                                                  J 

ui^   i  ■■'   ■■  t 

4-A                            / 

^   V   t  ■ 

1     ^  r :        & 

'■=  5                 -4i 

f  /                       T 

Z              'x-^            M 

J 

^%  f  ^^  ¥- 

Z                          2 

.0         J^.J^I-\Wl 

% 

-■■■   /  ■-A\3£    3lE 

VOON  I       234-56789      10      II    MT.     I       23*56       739      10     II    WON  I 

Fig.  42. — Series  20,  showing  movement  in  upper  (A)  and  lower  (B)  stomata  of 
heavily  watered  potato  plants,  and  in  upper  (C)  and  lower  (D) 
stomata  of  plants  in  vei-y  dry  soil. 

Since  the  stomatal  movement  of  the  cut  stems  was  very  different 
from  that  of  the  other  plants,  it  is  clear  that  the  water-loss  shown  by 
the  potometers  can  not  be  considered  representative  of  the  trans- 
piration from  any  field  plants  or  watered  potted  plant.  Moreover, 
because  of  the  difference  found  in  the  stomatal  movement  of  heavily 
watered  potted  and  field  plants,  the  transpiration  from  a  plant  in 
a  sealed  pot  can  not,  under  these  conditions,  be  considered  to  be 
the  same  as  that  from  a  field  plant.  The  plants,  however,  were  under 
conditions  of  high  evaporation,  high  temperature,  and  very  high 
radiation.  The  potted  plants  were  influenced  by  the  increased 
temperature  of  the  soil-mass,  due  to  the  action  of  radiant  heat. 
The  effect  of  extreme  insolation  is  shown  especially  by  the  cut  stems, 
including  those  in  the  potometers,  which  did  not  recover  turgor 
during  the  night.  Similar  potometers  kept  in  the  shade  functioned 
a  week  before  being  seriously  affected  by  bacterial  action.  This 
experiment  and  the  care  taken  to  prevent  bacterial  infection  of  the 
stems  and  the  consequent  occlusion  of  the  vessels  tend  to  show  that 
wilting  was  not  due  to  this  cause. 

As  in  the  case  of  alfalfa,  the  stomatal  openings  found  on  the  upper 
and  lower  surfaces  of  the  leaves  were  averaged  in  proportion  to  the 
number  of  stomata  present  per  unit  area  for  comparison  with  the 
rate  of  transpiration.     The  changes  found  in  one  correspond  fairly 


88 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 


well  with  those  found  in  the  other,  especially  when  the  probable 
effect  of  sunlight  and  relative  humidity  are  taken  into  consideration. 
The  agreement,  however,  is  not  so  close  as  in  the  alfalfa  experiment 
for  several  reasons,  in  addition  to  those  already  enumerated.  Later 
experiments  as  well  indicate  that  changes  in  the  degree  of  stomatal 
opening  are  not  nearly  so  effective  in  producing  changes  in  trans- 
piration when  near  the  maximum  opening  as  when  approaching 
closure.  Moreover,  in  this  as  well  as  in  other  experiments,  the 
stomata  of  the  two  surfaces  do  not  have  an  equal  effect  upon  the 
control  of  transpiration  in  a  leaf  having  unlike  stomata  upon  the 
upper  and  lower  surfaces.  Hence,  the  rate  of  water-loss  corresponds 
more  nearly  at  times  to  the  movement  found  in  the  lower  surface 
than  to  the  average  stomatal  movement.  As  the  ratio  of  upper 
stomata  to  lower  was  3  :  20  per  unit  area  in  this  variety,  the  effect 
of  the  upper  stomata  upon  the  calculated  average  was  not  large. 
If,  in  addition  to  the  use  of  this  ratio,  all  changes  of  opening  above 
50  per  cent  of  the  maximum  had  been  ignored,  the  effectiveness  of 
stomatal  movement  upon  transpiration  would  probably  have  been 
shown  with  greater  accuracy  (fig.  43) . 

The  water-loss  from  noon  to  1  p.  m.  on  August  25  was  91.6  mg. 
per  minute.  Stomatal  opening  during  this  time  changed  from  100 
to  91  per  cent.     A  sudden  reduction  to  15  per  cent  in  the  lower 


\ 

!  ! 

\ 

\ 

\\ 

^'\ 

^> 

^ 

^ 

B 

V 

\ 

s 

^ 

y 

V 

,- 

-_ 

\ 

/ 

\ 

\^ 

.^ 

,"" 

-" 

'v 

— 

!~_ 

17_ 

iz; 

— 

^ 

— 

^ 

— 

;^ 

5       6       7      8       9      10      II    MT.     I        2       3 


5       6       7       8       9      to 


FiQ.  43. — Series  20,  showing  average  stomatal  movement  (A)  and  transpiration  (B)  in  cut 
stems  of  potato. 


stomata  at  2  p.  m.  brought  average  opening  to  26  per  cent  and  de- 
creased the  water-loss  to  52.5  mg.  The  next  hour  the  lower  stomata 
opened  to  25  per  cent,  while  the  average  of  opening  rose  only  to 
28  per  cent,  since  the  upper  stomata  closed  50  per  cent.  The  trans- 
piration-rate rose  to  64.7  mg.,  which  would  indicate  that  the  change 
from  maximum  to  half  opening  in  the  upper  stomata  did  not  have 
nearly  the  effect  upon  the  rate  of  water-loss  that  it  had  upon  the 
calculated  average  stomatal  movement.  At  4  p.  m.  the  average 
dropped  to  22.6  per  cent,  while  during  the  hour  transpiration  fell 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION.        89 

to  55.4  mg.  per  minute.  Closure  to  11  per  cent  was  accompanied 
by  a  fall  in  the  rate  to  35.9  mg.  per  minute.  The  stomata  then  opened 
to  20  per  cent  in  both  surfaces,  and  the  rate  rose  to  47.8  mg.  Closure 
to  12  per  cent  at  7  caused  a  falling  off  to  36.4  mg.  From  this  time 
there  was  a  slow  closure  of  stomata  and  a  very  slow  falling  off  in  the 
transpiration-rate.  A  slight  increase  in  opening  from  2.6  per  cent 
to  4.3  per  cent  at  midnight  probably  caused  the  shght  increase  in 
the  rate  of  water-loss  from  8.5  mg.  to  8.8  mg.  at  midnight.  At  5 
a.  m.  all  the  stomata  were  closed,  and  the  minimum  rate  of  7.2  mg. 
was  recorded  at  that  hour.  At  7  a.  m.  the  stomata  opened  3  per  cent 
and  the  rate  rose  to  12.6  mg.,  due  in  part  to  the  decrease  in  relative 
humidity.  At  8  a.  m.  average  opening  increased  to  5  per  cent  and 
the  rate  increased  to  15  mg.  At  9  a.  m.  the  average  increased  to  7 
per  cent,  the  maximum  for  the  morning,  while  the  rate  increased 
to  20.1  mg.  per  minute.  Between  9  and  10  a.  m.  the  stomata  closed 
from  7  to  3.5  per  cent,  but  because  of  the  rapid  fall  in  humidity  and 
of  increased  sunlight,  the  rate  rose  slightly,  reaching  21.1  mg.  per 
minute.  Reduction  to  3  per  cent  average  opening  at  11  a.  m,  brought 
on  a  slight  decrease  of  water-loss  to  21.0  mg.  The  stomata  remained 
in  this  condition  another  hour,  but  the  rapid  fall  of  relative  humidity 
caused  an  increase  in  the  rate  to  23.9  mg.  Closure  of  the  stomata  at 
1  p.  m.  brought  on  a  reduction  in  the  rate  to  21.7  mg.  per  minute 
(fig.  43). 

The  two  series  described  show  that  the  potometer  was  useless  as 
an  indicator  of  the  water-loss  from  a  field  plant,  or  even  a  potted 
plant  of  alfalfa  or  potato.  One  series  with  apple  and  peach  twigs 
in  the  potometers  indicated  that,  with  the  variety  of  apple  used  the 
water-loss  from  a  twig  may  represent  fairly  well  the  transpiration  of 
the  tree,  since  the  stomata  of  the  cut  twigs  and  of  the  tree  cor- 
responded to  some  extent;  but  even  in  this  case  there  was  some 
divergence.  The  great  lack  of  correlation  between  transpiration  and 
stomatal  movement  in  Verbena  ciliata  and  Fouquiera  splendens  as 
found  by  Lloyd  supported  the  view  that  no  more  correlation  existed 
between  the  stomatal  movement  of  cut  stems  and  rooted  plants  in 
the  case  of  these  plants  than  in  alfalfa  or  potato.  Hence,  when 
the  opportunity  arose  to  test  this  view  directly,  a  series  was  made  with 
these  plants. 

This  series,  carried  out  at  the  Desert  Laboratory,  Tucson,  Arizona, 
was  begun  at  9  a.  m.  on  March  15,  1918,  and  ended  9  a.  m.  the  next 
day.  It  was  conducted  in  the  same  manner  as  the  alfalfa  series, 
the  water-loss  from  potometers  being  measured  at  hour  intervals, 
and  sets  of  epiderm  collected  from  cut  stems,  and  watered  and 
unwatered  field  plants.  The  day  was  fairly  warm,  clear,  and  very 
dry.  The  humidity  varied  from  12  per  cent  at  3  p.  m.  to  a  maximum 
of  47  per  cent  at  4  a.  m.  The  temperature  varied  from  88°  F.  at  noon 
to  50.5°  F.  at  4^30'""  a.  m.     The  sunlight  curve  was  rather  regular, 


90        EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 

though  a  morning  haze  prevented  maximum  from  being  reached 
until  1  p.  m.  (fig.  44). 

The  stomata  of  the  field  plants  of  Fouquiera  were  wide  open  at 
the  start  of  the  series  and  remained  open  until  noon.  At  1  p.  m. 
they  closed  to  85  per  cent  and  continued  until  20  per  cent  was  reached 
at  4  p.  m.    No  further  change  took  place  until  midnight,  when  they 


=" 

— 

s 

~1 

n 

~~1 

/ 

/ 

^^ 

■ — 

N 

/ 

^ 

V 

\ 

/ 

\ 

V. 

La 

— 

B 

^ 

^ 

' 

~1 

u 

,-- 

— 

- 

yi 

\ 

p- 

'' 

\\ 

— 

^ 

"^ 

\ 

,' 

■' 

1 

^, 

-- 

• — 1 

.,- 

-A 

1 

\ 

^ 

1 

5       6        7       8       9 


5      e       7      8       9      10 


Fig.  44. — Series  29,  weather  data  for  March  15-16,  1918;  sunlight  (A),  tempera- 
ture (B),  humidity  (C). 

closed  to  10  per  cent.    At  1  a.  m.  they  opened  to  20  per  cent  and  at 

2  to  25  per  cent.  They  remained  in  this  condition  an  hour  and  then 
closed  to  5  per  cent  at  4  a.  m.  At  5  a.  m.  they  opened  to  20  per  cent, 
35  per  cent  at  6,  50  per  cent  at  7,  and  80  per  cent  at  8  a.  m.  At 
9  a.  m.  they  were  again  wide  open.  Watering  had  no  effect  upon  the 
stomatal  movement  exhibited.  This  movement  is  apparently  in 
full  accord  with  that  found  by  Lloyd  for  this  species. 

The  stomatal  movement  found  in  the  cut  stems  of  this  plant, 
however,  differed  greatly.  Unlike  the  cut  stems  of  alfalfa  and  potato, 
there  was  no  complete  closure.  At  no  time  did  the  stomata  show 
less  than  65  per  cent  opening.  As  in  the  field  plants,  the  stomata 
were  wide  open  at  the  start  and  remained  at  maximum  until  1  p.  m., 
when  they  gradually  began  to  close.  At  4  p.  m.  they  were  70  per 
cent  open  and  then  reopened.  At  5  they  were  80  per  cent  open,  at 
6,  90  per  cent,  and  at  7  at  maximum  opening  again.  At  8  p.  m. 
they  closed  to  90  per  cent,  remained  in  this  condition  2  hours,  and 
at  1 1  p.  m.  closed  to  75  per  cent.  At  midnight  they  were  70  per  cent 
open,  at  1  a.  m.  65  per  cent,  and  so  remained  until  4  a.  m.  At  5  a.  m. 
they  reopened  to  75  per  cent,  at  6  to  85  per  cent,  at  7  to  90  per  cent, 
and  to  maximum  at  8  a.  m.  They  were  still  wide  open  when  the 
series  closed  (fig.  45). 

The  behavior  of  Yerhena  dliata  was  just  as  striking.  As  in  Fou- 
quiera, the  stomata  of  the  field  plants  were  wide  open  at  the  start 
of  the  experiment  and  remained  wide  open  until  noon.  They  then 
started  to  close,  the  process  being  rather  rapid  and  uniform.     At 

3  p.  m.  they  were  closed  to  15  per  cent.     After  this  the  rate  of  closure 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 


91 


became  slow,  the  stomata  being  12  per  cent  open  at  4  p.  m.,  10  per 
cent  at  5  p.  m.,  8  per  cent  at  6,  6  per  cent  at  7,  and  3  per  cent  at 
8  p.  m.  At  9  p.  m.  closure  was  complete  and  the  stomata  remained 
closed  until  after  1  a.  m.  At  2  a.  m.  they  opened  5  per  cent  and  at 
3  to  12  per  cent.  They  remained  in  this  condition  until  6  a.  m.  when 
they  opened  30  per  cent.  At  7  a.  m.  they  were  50  per  cent  open,  at 
8  a.  m.  they  were  80  per  cent,  and  at  9  a.  m.  they  were  again  wide 
open.  As  with  Fouquiera,  watering  some  of  the  plants  caused  no 
change  in  stomatal  movement.  The  probable  reason  was  that 
all  the  plants  had  sufficient  moisture,  and  the  amount  added  did  not 
result  in  enough  increase  to  produce  a  visible  change.  This  was  not 
checked  by  sampling  the  soil;  hence  it  can  only  be  inferred. 

As  in  Fouquiera,  the  cut  stems  of  Verbena  showed  no  complete 
closure  of  the  stomata.  They  were  wide  open  at  the  start  of  the 
experiment  and,  like  the  rooted  plants,  remained  open  until  noon.    At 


n 

^ 

^ 

/ 

\ 

/ 

7 

so 

70 

\ 

k 

\ 

/ 

\ 

^ 

-^ 

/ 

\ 

\ 

/ 

N 

^ 

/ 

/ 

> 

^ 

/ 

50 

\ 

/ 

lU 

\ 

t 

r 

£0 

> 

A 

^ 

— 

L 

/ 

\ 

y 

\ 

/ 

f 

__ 

_ 

10      II    NOON  I       Z 


34-56789       10      II   MT.      I        Z        3456        7,1910 


FiQ.  45. — Series  29,  showing  average  "movement  in''upper  and  lower  stomata 
of  watered  and  unwatered  field  plants  (A)  and  cut  stems  (B)  of 
Fouquiera  splendens. 


oo 

"■" 

.... 

' 

/ 

7 

■"" 

\ 

\ 

/ 

f 

00 

^ 

, 

\ 

/ 

\ 

f 

I 

63 

\ 

\ 

/ 

\ 

/ 

/ 

\ 

^ 

^ 

/ 

/ 

^ 

/ 

/ 

f 

/ 

».^ 

f 

~^ 

' 

^ 

^ 

y 

10     II  NOON  1       234       56       789      10     II    MT.     I       E3456       789      10 


FiQ.  46. — Series  29,  showing  average  movement  in  upper  and  lower  stomata  of 
field  plants  (A)  and  cut  stems  (B)  of  Verbena  ciliata. 

1  p.  m.  they  closed  to  90  per  cent,  at  2  to  80  per  cent,  and  to  70  per 
cent  at  3  p.  m.  They  remained  in  this  condition  until  5  p.  m.,  and 
then  closed  to  60  per  cent  at  6.  They  closed  to  55  per  cent  at  7  p.  m., 
50  per  cent  at  8,  and  45  per  cent  at  9  p.  m.    No  further  change  oc- 


92 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 


curred  until  after  1  a.  m.  At  2  a.  m.  they  opened  to  60  per  cent,  to 
70  per  cent  at  3,  and  to  80  per  cent  at  4  a.  m.  At  5  a.  m.  they  had 
closed  to  55  per  cent,  but  opened  to  70  per  cent  at  6,  and  to  90  per 
cent  at  7  a.  m.  At  8  a.  m.  they  were  wide  open,  and  remained  open 
to  the  end  of  the  series  (fig.  46). 


r 

A 

s 

.. 

/ 

-^ 

r\ 

' 

\ 

\ 

\ 

/ 

\ 

A 

/ 

/ 

\ 

/ 

\ 

/ 

< 

/ 

\ 

A 

\ 

-V 

/ 

/ 

\ 



— 

y 

/ 

\ 

r^ 

\ 

V 

^ 

^ 

'N 

^ 

/ 

\. 

— 

/ 

^ 

--. 

-^ 

V 

Fig  47. — Series  29,  showing  stomatal  movement    (A)    and    transpiration 
(B)  in  cut  stems  of  Fouquiera  splendens. 

There  was  no  close  correlation  between  stomatal  movement  and 
transpiration  in  either  species.  The  maximum  transpiration-rate 
for  Fouquiera  occurred  between  noon  and  1  p.  m.,  and  was  clearly 
the  result  of  low  humidity  and  maximum  sunlight  at  the  time  when 
the  stomata  were  still  open.  After  this  time  the  rate  fell,  in  spite  of 
the  fact  that  the  light  was  still  at  maximum  and  humidity  less,  owing 
to  partial  closure  of  the  stomata.  The  fall  was  not  rapid,  however, 
until  between  4  and  6  p.  m.,  when  the  humidity  rose  somewhat  and 
sunhght  fell  off  rapidly.  But  as  the  stomata  at  no  time  closed  to 
less  than  65  per  cent,  their  effect  upon  the  water-loss  of  the  plant 
was  mostly  overshadowed  by  that  of  humidity  and  sunlight,  as  well 
as  air-temperature  and  wind  (fig.  47). 

In  Verbena  the  maximum  rate  of  transpiration  was  reached  be- 
tween 11  a.  m.  and  noon,  an  hour  earher  than  in  Fouquiera.  The 
rate  was  only  slightly  less  on  the  following  hour,  as  there  was  but 
little  closure.  The  rapid  fall  in  the  rate  occurred  at  the  same  time 
as  in  Fouquiera  and  was  clearly  the  result  of  changes  in  the  physi- 
cal factors  and  not  of  stomatal  movement.  Just  as  in  Fouquiera, 
the  fact  that  the  stomata  of  cut  stems  never  closed  sufficiently  to 
cause  close  regulation  of  transpiration  allowed  the  water-loss  to 
be  controlled  almost  completely  by  the  factors  of  evaporation  (fig.  48). 

Throughout  his  investigation,  Lloyd  (1908)  apparently  made  no 
parallel  series  of  observations  upon  the  stomatal  movement  in  cut 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION.        93 

stems  and  rooted  plants.  But  in  a  later  paper  (1912),  he  checked 
the  rate  of  loss  against  the  rate  of  absorption  of  water  in  the  cut 
stem  of  Fouquiera  and  at  the  same  time  apparently  measured  the 
stomatal  movement  in  one  of  the  cut  stems.  As  a  result,  the  stomatal 
movement  illustrated  is  like  that  found  here  to  be  typical  of  the 
movement  in  cut  stems  of  Fouquiera.  Throughout  the  period 
shown  in  his  graph  the  stomata  fluctuated  between  50  and  90  per 
cent  of  maximum,  at  no  time  closing  to  a  point  where  they  would 
closely  regulate  the  water-loss  from  the  potometer.  He  states,  how- 
ever, that  the  stomata  were  2.4  microns  open  at  midnight  preceding  the 
experiment,  but  evidently  the  stem  had  just  been  removed,  since  in 
none  of  the  present  series  have  the  stomata  of  a  cut  stem  of  Fouquiera 
closed  to  this  degree,  i.  e.,  28  per  cent.  Regarding  the  failure  of  the 
stomata  to  close  during  the  afternoon,  he  states:  "the  behavior  dur- 
ing the  latter  part  of  the  day  can  not  with  certainty  be  regarded  as 
wholly  normal,  as  it  does  not  accord  with  my  previous  results." 


- 

r~ 

-> 

1  ! 

[ 

\ 

1 

\ 

; 

/ 

B 

\ 

\ 

/ 

/ 

\ 

\ 

/ 

\ 

A 

\ 

/ 

\ 

t 

,u 

/ 

\^ 

/ 

\ 

/ 

/ 

V 

■v 

^ 

/ 

\ 

r 

" 

\_ 

*x 

/ 

l\ 

1 

/ 

- 

' 

s. 

{ 

V 

^ 

N 

J 

- 

.u 

-v^ 

Fig.  48. — Series  29,  showing  stomatal  movement  (A)  and  transpiration 
(B)  in  cut  stems  of   Verbena  ciliata. 


Knight's  evidence  against  stomatal  regulation  is  largely  based  upon 
"relative  transpiration"  readings  as  compared  with  the  stomatal 
aperture  index  as  measured  by  the  porometer.  As  previously  stated, 
it  is  to  be  doubted  whether  anyone  really  knows  what  this  index 
represents,  since  apparently  no  one  has  compared  it  with  direct 
observations  of  the  stomatal  apertures  which  it  is  supposed  to  meas- 
ure. Moreover,  "relative  transpiration"  is  meaningless  until  some 
instrument  is  devised  that  will  represent  with  some  degree  of  accuracy 
the  effect  upon  the  plant  of  each  factor  concerned  in  evaporation. 
This  objection  applies  to  Trelease  and  Livingston's  investigation 
(1916)  as  well. 


94        EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 

The  relationship  between  the  water-loss  and  stomatal  movement 
of  heavily  watered  plants  on  the  verge  of  wilting  from  lack  of  water, 
and  cut  stems,  was  investigated  in  alfalfa  series  32,  begun  7  p.  m. 
on  August  25  and  ending  7  p.  m.  August  26,  1919.  The  night  was 
clear  and  starlit,  no  moon  showing.  During  the  first  part  of  the 
evening  there  was  no  wind,  but  one  rose  toward  midnight,  char- 
acterized by  short  gusts  and  intervals  of  calm.     Between  12  and 

I  a.  m.  it  rose  to  a  maximum  of  3,700  feet  for  the  hour,  and  then 
slowly  died  down,  disappearing  between  3  and  4  a.  m.  Relative 
humidity  was  not  high,  but  rather  variable,  the  mean  rising  from  38 
per  cent  at  7  p.  m.  to  a  maximum  of  63  per  cent  at  3  a.  m.  and  again 
at  7  a.  m.    The  temperature  fell  from  79°  F.  at  7  p.  m.  to  63.5  at 

II  p.  m.,  rose  again  to  70°  F.  at  1  a.  m.  and  then  fell  again  to  64°  F. 
at  3  and  4  a.  m.  The  next  day  was  slightly  hazy  and  with  large 
drifting  clouds,  which  four  times  during  the  day  reduced  the  sun- 
light. Although  the  sun  rose  before  6  a.  m.,  it  was  not  until  after 
7  a.  m.  that  the  sunlight  reached  10  per  cent.  The  maximum  for  the 
day  was  reached  at  2*" SO""  p.  m.,  when  the  light  was  85  per  cent. 
Sunset  occurred  shortly  after  the  series  ended.  The  maximum  tem- 
perature was  89°  F.  at  2  p.  m. ;  hence  the  day  was  very  warm.  Be- 
tween 8  and  9  a.  m.  the  wind  rose  suddenly  to  12,200  feet  for  the 
hour,  fell  to  9,800  feet  the  next  hour,  and  then  averaged  3,000  feet 
per  hour  during  the  rest  of  the  day,  dying  away  at  sunset.  During 
the  period  the  barometric  pressure  fluctuated  from  25.4  inches  to 
25.7  inches  from  7  p.  m.  on  the  25th  to  noon  the  next  day  (fig.  49). 


120 
110 

80 
70 

'■<-' 

\ 

^ 

^ 

N 

N 

/ 

f 

--J 

\ 

-H 

y 

^ 

— 

B 

— 

h: 

r 

60 
50 
4-0 
30 
20 
10 

,'' 

\ 

^'' 

""■ 

--' 

•c 

^' 

V 

<■ 

•'' 

;\ 

—  r 

\ 

/' 

'\ 

i 

V 

•-. 

,•■ 

0/ 

y 

^ 

/ 

-— 

■— 

A 

■■ 

y 

/ 

\ 

\ 

^ 

/ 

1 

\ 

. 

234-       56789      10 


Fig.  49. — Series  32,  weather  data  for  August  25-26,  1919;  sunlight  (A), 
temperature  (B),  humidity  (C),  wind  velocity  (D). 

The  water-loss  from  heavily  watered  plants  was  determined  by 
weighing  small  plants  in  metallic  containers  at  2-hour  intervals 
upon  a  precision  balance,  weighing  them  to  the  nearest  milligram. 
The  total  weight  of  each  plant  and  container  was  approximately  150 
grams.    The  plants  stripped  were  somewhat  larger,  but  of  the  same 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 


95 


age,  and  were  grown  in  large  earthenware  pots,  an  average  of  10 
plants  to  a  pot.  Great  care  was  exercised  to  keep  the  soil  tempera- 
ture of  the  pots  and  containers  the  same  throughout  the  day,  and 
the  plants  under  the  same  conditions  in  other  respects.  This  was 
done  by  embedding  the  metal  containers  in  the  soil  of  the  pots,  and 
keeping  the  soil  from  the  container  by  a  metal  cyUnder.  A  small 
piece  of  moist  flannel  was  kept  over  the  top  of  the  container,  except 
when  weighed,  in  order  to  duplicate  as  closely  as  possible  the  moist 
soil-surface  at  the  base  of  the  other  plants.  The  containers  were 
removed  for  about  5  minutes  every  2  hours  for  the  purpose  of  weigh- 
ing. They  were  weighed  on  the  half  hour,  in  order  that  the  interval 
would  coincide  better  with  the  stomatal  observations.     Strips  were 


-rr 

_ 

/ 

N. 

/ 

/ 

\ 

~ 

r- 

— 

- 

lOU 

/" 

' 

/ 

/ 

} 

- 

1 — ' 

t\ 

/ 

^1 

/b 

V 

/ 

\ 

- 

— r 

_ 

h 

f- 

- 

\ 

s 

1 

/ 

\ 

\ 

J 

/ 

\ 

\ 

\ 

I 

Js 

_ 

\; 

s- 

^ 

J 

Q      9      10     II    MT. 


3      4-56 


2      3      4       5      6 


Fig.  50. — Series  32,  showing  stomatal  movement  averaged  for  2-hour  periods 
(A)  and  transpiration  in  milligrams  per  minute  for  the  same 
periods  (B)  in  heavily  watered  potted  plants  of  alfalfa. 

collected  at  hour  intervals,  but,  for  purposes  of  comparison,  the  two 
sets  of  strips  collected  during  each  interval  between  weighings  was 
averaged.  This  method,  of  course,  produced  smoothed  curves,  but  as 
it  was  not  possible  to  weigh  at  shorter  intervals,  the  smaller  fluctua- 
tions could  not  be  taken  into  account. 

From  7  to  S'^SO"'  p.  m.  the  water-loss  of  the  watered  plants  was 
44.7  mg.  per  minute  per  square  decimeter  of  leaf-surface.  During 
this  period  the  stomatal  opening  was  16  per  cent.  From  8''30'"  a.  m. 
to  10''30™  p.  m.  the  loss  was  11.4  mg.  per  minute  and  the  average 
stomatal  opening  3  per  cent.  The  same  rate  of  loss  occurred  during 
the  next  2-hour  period,  while  the  stomatal  opening  was  4  per  cent. 
The  lack  of  a  corresponding  increase  is  accounted  for  by  the  rise  of 
relative  humidity  and  the  fall  in  the  rate  of  evaporation  shown  by 
the  two  types  of  atmometer.  During  the  next  period,  from  12^30°"  to 
2''30"'  a.  m.,  the  average  opening  was  again  3  per  cent,  but  as  the 
rate  of  evaporation  rose  again,  the  rate  of  water-loss  remained  at 


96        EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 

11.4  mg.  as  before.     During  the  period  between  2''30'"  and  4^*30™ 
a.  m.  the  average  of  stomatal  opening  rose  to  6  per  cent,  but  as  the 
humidity  increased  to  the  maximum  and  evaporation  fell  off  to  the 
minimum,  the  rate  of  transpiration  fell  slightly,  namely,  to  10.2 
mg.  per  minute.    During  the  next  period,  4''30""  to  6^'30'"  a.  m.,  the 
average  of  stomatal  opening  rose  to  25  per  cent,  but  as  evaporation 
and  relative  humidity  remained  the  same,  this  in  part  explains  the 
slight  increase  to  12.4  mg.     During  the  period  from  6''30"'  to  8^30"' 
a.  m.  the  average  rose  to  85  per  cent  and  the  rate  of  water-loss  to 
48.3  mg.     Evaporation  increased  somewhat  during  this  period,  but 
was  still  very  low.    The  average  opening  rose  to  100  per  cent  during 
the  period  from  8^30"'  to  10^30""  a.  m.  and  the  rate  of  loss  to  80  mg. 
per  minute.     The  stomata  were  wide  open  during  the  following 
period  as  well,  while  the  rate  further  increased  to  108  mg.  per  minute 
in  response  to  increase  of  evaporation.     From  12''30'"  to  2''30"'  p.  m. 
the  evaporation  was  very  high  comparatively  and  the  transpiration- 
rate  rose  to  the  maximum  of  132.7  mg.  per  minute,  the  stomata  still 
being  wide  open.     During  the  interval  from  2''30™  to  4''30"'  p.  m. 
the  rate  fell  to  106.7  mg.,  largely  in  response  to  a  similar  fall  in  the 
intensity  of  the  factors  of  evaporation,  although  at  the  end  of  the 
period  the  stomata  closed  sHghtly.     From  4''30"'  to  6''30"'  p.  m.  the 
rate  fell  to  56.8  mg.  per  minute,  in  part  due  to  the  fall  of  evaporation 
and  in  part  to  closure  of  the  stomata,  the  average  opening  for  the 
period  being  62  per  cent.     The  fluctuations  of  the  rate  while  the 
stomata  were  wide  open  are  clearly  caused  by  the  changes  in  the 
evaporating  factors,  and  it  is  evident  that  these  will  control  transpira- 
tion while  the  stomata  are  at  or  near  their  maximum  opening  (fig,  50). 
The  plants  in  the  dr\"  containers  were  treated  in  the  same  manner 
as  the  preceding  ones.    The  plants  stripped  were  small  ones  in  large 
-earthenware  pots,  and  differed  from  those  heavily  watered  in  having 
had  no  water  for  10  days.     The  water-content  for  this  period  was 
10  per  cent,  and  the  echard  was  9  per  cent.     The  containers  were 
weighed,  as  before,  but  alternating  with  the  watered  plants.     On 
this  account  the  intervals  overlap  those  for  the  other  set  by  1  hour, 
and  hence  the  curves  alone  can  be  compared  for  the  two. 

As  before,  the  stomatal  movement  was  averaged  for  the  2  hours 
of  the  interval  between  weighings.  Between  7''30'"  and  8*^30  p.  m. 
this  average  was  2  per  cent,  while  the  rate  of  transpiration  was  7.91 
mg.  per  minute.  Between  9''30"'  and  ll''30"'  p.  m.  the  rate  fell  to 
5*^40"'  mg.  per  minute,  in  spite  of  the  slight  increase  of  opening  to 
3  per  cent.  This  fall  coincides  with  a  great  reduction  in  the  rate 
of  evaporation  as  shown  by  the  atmometers,  the  average  for  the 
first  period  being  195  mg.  per  minute  and  for  the  second  46  mg.  per 
minute.  In  the  interval  from  11^30"'  to  1^30"'  a.  m.  the  rate  was 
practically  unchanged,  being  5.42  mg.  per  minute,  although  the 
stomata  showed  again  a  slight  opening.    From  1^30"'  to  3''30°'  a.  m. 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION.        97 

the  average  stomatal  opening  increased  to  25  per  cent,  while  the 
rate  rose  to  8.73  mg.  per  minute.  A  sUght  increase  in  opening  to 
28  per  cent  from  S'^SO"'  to  5^30™  a.  m.  corresponded  to  slight  increase 
in  the  rate  to  8.75  mg.,  although  the  rate  of  evaporation  decreased. 
The  maximum  of  opening  occurred  from  5^30"'  to  7''30"'  a.  m.,  the 
average  being  58  per  cent,  and  the  greatest  transpiration  also  oc- 
curred at  this  time,  the  water-loss  being  24.21  mg.  per  minute. 
Closure  to  8  per  cent  from  7^30"'  to  9''30"'  a.  m.  caused  in  part  the 
drop  in  the  rate  to  4.75  mg.,  but  the  lowest  rate  of  evaporation 
observed  was  partly  also  responsible.  From  9^'30'"  to  ll^'SO'"  a.  m. 
the  average  opening  decreased  to  6  per  cent,  but  the  rise  in  evapora- 
tion to  three  times  its  former  rate  caused  a  slight  increase  in  the 
rate  of  transpiration  to  5.11  mg.  per  minute.  From  11^30""  a.  m. 
to  l^SO""  p.  m.  the  average  opening  and  evaporation  were  unchanged, 
and  the  rate  of  water-loss  also  remained  the  same.  From  1^30°'  to 
3^30  p.  m.  the  stomata  were  again  unchanged,  but  the  great  increase 
of  evaporation  from  85  mg.  to  268  mg.  per  minute  caused  a  rise  in  the 
rate  of  transpiration  to  10.67  mg.  per  minute.  During  the  period 
from  3^'30™  to  5^30'"  p.  m.  the  stomata  opened  to  8  per  cent,  and 
although  the  evaporation-rate  dropped  considerably,  the  rate  of 
transpiration  rose  to  10.75  mg.  per  minute.     From  5''30"'  to  7''20"' 


t 

/ 

\ 

i 

\ 

no 

/ 

— 

\ 

/ 

\ 

/ 

\ 

/ 

I 

\ 

/ 

\ 

\ 

\ 

'  \ 

/ 

\ 

\ 

\ 

\ 

\ 

^ 

\ 

V 

/ 

\ 

y^ 

\ 

N^ 

V 

^ 

^ 

\ 

t 

\ 

__^ 

^ 

•-' 

'v, 

^ 

/ 

. 

B 

/ 

/ 

\ 

B 

\= 

_ 

10 

-- 

==. 

— 

=- 

-i 

=- 

._ 

B 

_ 

- 

:-- 

■"* 

— 

'£] 

_ 

zz 

^ 

3     4-56 


3      4      5      6      7 


Fig.  51. — Series  32,  showing  stomatal  movement  averaged  per  2-hour  period 
(A)  and  transpiration  in  milligrams  per  minute  (B)  of  dry  alfalfa 
phytometers;  evaporation  1=2  milligrams  per  minute  (C). 

the  stomata  closed  to  4  per  cent  and  the  rate  of  evaporation  fell 
below  100  mg.  per  minute,  and,  in  consequence,  the  average  water- 
loss  fell  to  5.06  mg.  per  minute.  While  this  shows  that  the  factors 
of  evaporation  play  an  important  part  in  changing  the  rate  of  trans- 
piration, the  effect  of  these  factors  is  as  clearly  shown  to  be  regulated 
>by  the  stomata  (fig.  51). 


98 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 


A  comparison  of  the  transpiration-rates  of  the  two  groups  of 
plants  brings  out  certain  other  interesting  facts.  At  only  one  time 
did  the  rate  for  the  plants  sealed  in  dry  soil  apparently  exceed  that 
of  the  heavily  watered  plants.  This  probably  happened  between  5 
and  6  a.  m.,  although  because  the  two  sets  were  weighed  at  alternate 
half  hours  it  was  not  actually  demonstrated.  This  was  due  to  the 
much  greater  stomatal  opening  at  that  time  in  the  plants  in  dry 
soil.  Another  point  of  importance  is  shown  by  the  comparative  rates 
in  the  periods  including  10  p.  m.  in  both  series.  At  this  time  the 
stomata  of  both  sets  averaged  3  per  cent  opening,  but  the  rate 
for  the  heavily  watered  plants  was  practically  twice  that  of  the  dry 
plants.  Throughout  the  experiment  the  rate  was  proportionally 
lower  for  the  plants  in  dry  soil  for  the  given  degree  of  stomatal 
opening.  The  most  plausible  explanation  is  to  ascribe  this  to  the 
effect  of  a  lower  water-margin  in  the  leaf.  Moreover,  it  is  very 
evident  that  the  much  lower  rate  of  water-loss  in  the  dry  containers 
during  the  greater  part  of  the  day,  when  the  rate  for  the  wet  ones  was 
very  high,  must  be  attributed  to  the  control  exerted  by  the  nearly 
closed  stomata  (fig.  52). 


/ 

/\ 

s 

no 

/ 

/ 

\ 

/ 

\ 

/ 

] 

DO 

/ 

\ 

/ 

\ 

70 

/ 

V 

y 

\ 

^0 

\ 

f 

V 

00 

\ 

10 

^ 

u 

^^ 

^ 

^ 

^ 

— 

— 

1 — 1 

r 

^ 

7      8      9      10     11    MT.     I       2      3      4-      5      6      7      8      9      10     1 1  NOON   I       2      3-*       5      6      7 

Fig.  52. — Scries  32,  showing  transpiration  of  wet  (A)  and  dry   (B)  alfalfa 
phytometers. 

The  stomatal  movement  of  plants  of  cow-beet  growing  in  large 
pots  of  dry  soil  was  studied  in  a  similar  manner  in  series  33.  This 
experiment  was  started  at  6  p.  m.  on  August  8,  1919,  and  ended  at 
6  p.  m.  on  August  9.  The  night  was  not  cold,  but  lowest  tempera- 
tures were  59°F.  at  2  a.  m.  and  59.5° F.  at  6  a.  m.,  just  before  sunrise. 
During  the  day  it  rose  to  a  maximum  of  85°  F.  at  2  and  3  p.  m.  The 
relative  humidity  fluctuated  a  great  deal,  but  within  unusually 
narrow  Umits,  since  it  did  not  rise  above  79  per  cent  or  fall  below 
42  per  cent.  Passing  clouds  caused  the  sunlight  to  fluctuate  con- 
tinuously. Like  the  other  factors,  the  wind  was  also  variable,  at 
times  rising  to  an  average  of  5  miles  an  hour  and  other  times  dying 
away  completely  (fig.  32). 


EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION.       99 

Two  weeks  before  the  experiment,  three  plants  were  transplanted 
into  the  metal  containers,  watered,  and  left  to  establish  themselves. 
Since  they  were  planted  in  soil  of  known  water-content,  and  weighed 
before  watering,  they  were  readily  brought  to  the  desired  water- 
content  at  the  start  of  the  experiment.  Twelve  large  plants  were 
also  placed  in  earthenware  pots  with  the  same  water-content.  Since 
the  low  water-content  of  the  dry  containers  of  the  alfalfa  series 
caused  more  closure  than  desired,  the  containers  in  this  case  were 
started  at  14  per  cent  and  fell  to  10  per  cent  at  the  end.  No  watering 
was  done  during  the  series,  since  this  tends  to  produce  localized 
regions  of  high  water-content.  The  containers  were  weighed  each 
hour,  immediately  after  collecting  the  strips. 

At  6  p.  m.,  when  the  series  started,  the  stomata  were  30  per  cent 
open,  and  10  per  cent  at  7  p.  m.  During  the  hour,  the  rate  of  water- 
loss  was  50  mg.  per  minute.  At  8  p.  m.  the  stomata  closed  to  1 
per  cent  and  the  rate  fell  to  11.25  mg.  At  9  p.  m.  the  stomata 
practically  closed,  a  few  slightly  open  in  the  upper  surface  making 
the  average  0.5  per  cent.  The  rate  of  water-loss  as  a  result  fell  to 
5.45  mg.  per  minute  during  the  hour.  At  10  p.  m.  the  stomata 
opened  to  an  average  of  5  per  cent  and  the  rate  rose  to  8.00  mg.  per 
minute.  At  1 1  p.  m.  further  opening  to  10  per  cent  occurred  and  the 
rate  rose  to  15.00  mg.  At  midnight  the  average  opening  decreased 
to  7  per  cent  and  the  rate  of  loss  decreased  to  10  mg.  per  minute. 
At  1  a.  m.  average  opening  decreased  to  1  per  cent,  but  a  great  in- 
crease of  evaporation  caused  a  shght  increase  of  water-loss  to  10.91 
mg.  As  the  stomata  continued  at  nearly  complete  closure  during 
the  following  hour,  the  rate  dropped  to4.62  mg.  per  minute.  At  3  a.  m. 
the  average  opening  increased  to  2.5  per  cent  and  the  water-loss  rose 
to  7.27  mg.  per  minute.  At  4  a.  m.  the  stomata  opened  to  10  per 
cent,  and  in  consequence  a  great  decrease  in  evaporation  caused 
but  a  slight  decrease  in  the  rate  of  water-loss  to  6.15  mg.  The  next 
hour  it  rose  to  10.91  mg.  per  minute.  From  5  to  6  a.  m.  the  stomata 
closed  from  3.0  per  cent  to  0.2  per  cent,  the  rate  of  evaporation  fell, 
and  hence  the  water-loss  fell  to  the  minimum  of  3.65  mg.  per  minute. 
The  lower  stomata  then  opened,  while  the  upper  remained  closed, 
bringing  the  average  to  32  per  cent.  As  evaporation  dropped  still 
lower,  the  increase  of  water-loss  was  only  11.67  mg.  The  stomata 
remained  at  this  opening  another  hour  and  the  rate  of  water-loss 
rose  to  41.43  mg.  per  minute.  Opening  to  75  per  cent  caused  a  rise 
to  52.73  mg.  per  minute,  and  to  a  maximum  of  120  nig.  on  the 
following  hour,  as  the  evaporation  rate  rose  very  much,  in  spite  of 
the  fact  that  the  stomata  began  to  close  agam.  However,  the 
greatest  water-loss  occurred  during  the  hour  the  stomata  were  at 
their  widest  opening  for  the  series.  Closure  to  15  per  cent  at  11  a.  m. 
caused  the  rate  to  decrease  to  78.33  mg.  The  stomata  opened  again 
to  18  per  cent  at  noon,  causing  the  rate  also  to  rise  to  83.33  mg. 


100     EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 

per  minute.  Further  opening  to  25  per  cent  at  1  p.  m.  resulted  in 
an  increase  of  water-loss  to  88.33  mg.  per  minute.  At  2  p.  m.  the 
stomata  closed  to  7  per  cent  and  the  rate  fell  to  66.67  mg.  per  minute. 
At  3  p.  m.  reopening  to  17  per  cent  was  accompanied  by  a  rise  in  the 
rate  to  72.73  mg.  per  minute.  From  this  time  to  the  end  of  the 
series  the  stomata  closed  slowly  and  the  rate  of  water-loss  fell  in 
proportion  (fig.  53). 


120 

~1 

_J  1 

K 

110 

A 

1 

v^ 

r\ 

\B 

^ 

\ 

\ 

\ 

V  1 

\ 

^ 

I 

^ 

L 

\ 

V 

^ 

1 

\ 

y 

s 

\y 

\ 

1 

) 

> 

\ 

/ 

\ 

s^ 

^ 

^ 

^ 

<. 

^ 

^ 

S 

^ 

L 

1 

■-«, 

** 

Fig.  53. — Series  33,  showing  average  movement  of  upper  and  lower  stomata 
(A)  and  transpiration  (B)  of  cow-beets  in  dry  pots. 

In  this  series,  as  well  as  in  the  alfalfa,  Fouquiera,  and  Verbena 
series,  direct  evidence  was  found  that  as  the  stomatal  movement 
of  cut  stems  and  rooted  plants  differed  considerably,  so  did  their 
rates  of  water-loss.  In  this  series  the  leaves  in  the  potometers 
showed  little  or  no  wilting,  but  their  stomata  were  closed  or  nearly 
closed  throughout  the  experiment.  At  the  start  the  stomata  were 
completely  closed  and  opened  to  7  per  cent  at  7  p.  m.  At  8  they 
closed  to  1  per  cent  and  at  9  were  completely  closed.  Thus  a  com- 
parison shows  that  the  stomatal  movement  of  the  cut  leaves  and 
the  plants  in  the  dry  pots  was  identical  at  8  and  9  p.  m.  and  differed 
not  at  all  in  the  rates  of  water-loss  at  this  time.  At  1  and  2  a.  m. 
the  stomata  of  the  potted  plants  were  practically  closed,  while  those 
of  the  cut  stems  were  2  per  cent  open;  hence  at  this  time  the  rate 
of  water-loss  for  the  cut  leaves  was  sUghtly  higher  than  that  of  the 
plants.  The  stomata  of  the  potted  plants  closed  again  at  5  and  6 
a.  m.,  while  the  cut  leaves  showed  4  per  cent  opening.  At  this  time 
the  transpiration-rate  of  the  potted  plants  again  fell  to  less  than  that 
of  the  cut  leaves.  At  all  other  times  the  transpiration-rate  of  the 
cut  stems  was  lower  and  during  the  day  considerably  lower  than 
that  of  the  potted  plants.  This  must  be  attributed  to  the  closed 
stomata  during  the  day  in  the  cut  leaves  (fig.  54). 

The  experiments  presented  show  that  stomata  regulate  the  water- 
loss  fiom  plants  and  contradict  the  results  of  Lloyd  (1908).  The 
reasons  for  this  lack  of  agreement  have  been  demonstrated,  and 


SUMMARY. 


101 


the  probable  causes  for  failure  of  agreement  with  certain  other 
investigations  have  been  indicated.  The  regulation  of  water-loss 
was  found  to  be  controlled  closely  by  the  stomata  when  they  are 
nearly  closed  and  by  the  factors  of  evaporation  when  they  are  wide 
open.  Brown  and  Escombe  (1900)  showed  that  diffusion  of  water- 
vapor  through  the  open  stomata  is  much  less  than  would  occur  if 


A 

\\ 

W 

f  \\V\\\ 

\ 

,/»S. 

\l 

V 

\ 

> 

' 

\ 

1 

{ 

,\^ 

^^ 

1 

B 

^._ 

^ 

<i 

.^ 

\^^^^ 

— 

-'•■' 

'~ 

~ 

Fig.  54. — Series  33,  showing  transpiration  from  potted  cow-beet  plants 
in  dry  soil  (A)  and  from  potometers  (B). 


the  air  within  the  leaf  were  saturated.  It  therefore  appears  prob- 
able that  the  air  is  saturated  only  in  the  deeper  spaces  of  the  leaf, 
and  that  as  the  outer  air  is  approached  through  the  passages,  sub- 
stomatal  chambers,  and  pores  of  the  stomata,  the  saturation  de- 
creases to  become  more  nearly  like  that  of  the  air  outside.  As  the 
stomata  close,  the  amount  of  saturated  intercellular  space  probably 
increases,  and  finally,  when  closure  becomes  complete,  all  the  air  con- 
tained in  the  leaf  becomes  saturated.  Therefore,  water-loss  is  most 
clearly  affected  by  changes  of  stomatal  opening  when  the  openings  are 
approaching  minimum. 

SUMMARY. 

1.  The  stomatal  movement  of  cut  stems  in  9  species  of  plants,  in- 
cluding Fouquiera  splendens  and  Verbena  ciliata,  differs  greatly  from 
that  found  in  potted  plants  and  field  plants,  except  in  the  one  exper- 
iment with  apple.    Even  in  this  case  there  was  some  divergence. 

2.  Since  the  stomatal  movement  is  different,  the  rate  of  water- 
loss  is  also  different  from  that  of  potted  plants  and  presumably  from 
field  plants. 

3.  Lloyd's  evidence  that  stomata  are  non-regulatorj'-  is  therefore 
vitiated  by  his  use  of  potometers  to  measure  the  water-loss  of  field 
plants. 

4.  Although  the  factors  concerned  in  evaporation  have  great 
influence  upon  transpiration,  this  influence  is  definitely  controlled 


102     EFFECT  OF  STOMATAL  MOVEMENT  UPON  TRANSPIRATION. 

by  the  stomata.  When  the  stomata  are  wide  open  or  nearly  wide 
open,  transpiration  is  the  result  of  the  action  of  the  factors  of  evapo- 
ration alone,  since  the  stomata  in  nowise  interfere  with  the  action. 
As  the  stomata  close,  the  influence  of  the  factors  is  lessened,  but 
until  closure  has  reduced  the  apertures  to  50  per  cent  or  less,  stomatal 
regulation  is  still  largely  overshadowed  by  the  control  exerted  by 
them.  When  closure  is  almost  complete,  the  regulation  of  water-loss 
by  the  stomata  is  very  close  and  the  effect  of  the  factors  over- 
shadowed by  the  effect  of  even  very  small  changes  of  the  opening. 


BIBLIOGRAPHY. 


Briggs,  L.  J.,  and  H.  L.  Shantz,  1916.     Hourly  transpiration  rate  on  clear  days  as  de- 
termined by  cyclic  environmental  factors.    Jour.  Agri.  Res.  5  :  583. 
.     1916.     A  daily  transpiration  during  the  normal  growth  period  and  its 

correlation  with  the  weather.     Jour.  Agri.  Res.  7  :  155. 
.     1917.     Comparison  of  the  hourly  evaporation  rate  of  atmometers  and 

free  water  surfaces  with  the  transpiration  of  Medicago  saliva.     Jour.  Agr. 

Res.  9:277. 
Brown,  H.  T.,  and  F.  Escombe.     1900.     Static  diffusion  of  gases  and  liquids  in  relation 

to  the  assimilation  of  carbon  and  translocation  in  plants.     Phil.  Trans. 

Roy.  Soc.  London  70  :  397. 
BuRGERSTEiN,  A.     1904.     Die  Transpiration  der  Pflanzen. 
BuscALiONi,  L.,  and  G.  Polacci.     1902.     Alteriori  ricerche  sull'  applicazione  delle  pel- 

licole  di  coUodio  alio  studio  di  alcuni  processi  fisiologici  delle  piante  ed  in 

particolar  modo  delle  traspirazione  vegetale.    Att.  1st.  Bot.  Univ.  Pavia. 

n.  s.  7: 
Clements,  F.  E.     1905.     Research  methods  in  ecology. 

.     1907.     Plant  physiology  and  ecology. 

Darwin,  Francis.     1898.    Observations  on  stomata.     Phil.  Trans.  Roy.  Soc.  London 

B  190  :  531. 
.     1914.     On    a   method   of  studying   transpiration.     Proc.    Roy.    Soc.    London 

B  87  :  269. 
.     1914.     The  effect  of  light  on  the  transpiration  of  leaves.     Proc.  Roy.  Soc. 

London  B  87  :  281. 

.     1916.     On  the   relation   between   transpiration   and   stomatal   aperture.     Phil. 

Trans.  Roy.  Soc.  London  B  207  :  413. 
and  D.  F.  M.  Pertz.     1911.     New  method  of  estimating  the  aperture  of  stomata. 

Proc.  Roy.  Soc.  London  B  84  :  149. 
Dixon,  H.  H.     1914.     Transpiration  and  the  ascent  of  sap  in  trees. 
EcKERSON,  Sophia  H.     1908.     The  number  and  size  of  stomata.     Bot.  Gaz.  46  :  221. 
Gray,  J.,  and  G.  J.  Peirce.     1919.     The  influence  of  hght  upon  the  action  of  the  stomata 

and  its  relation  to  the  transpiration  of  certain  grains.    Am.  Jour.  Bot.  6:131. 
Haberlandt,  G.     1904.     Physiologische  Pflanzenanatomie. 
Iljin,  W.  S.    1914.    Die  Regulierung  der  Spaltoffnungen  in  Zu-sammenhang  mit  Veriinder- 

ung  des  osmotischen  Druckes.     Beih.  Bot.  Cent.  32  :  15. 
.     1914  A.      Die  Probleme  des'  vergleichenden  Studiums  der  Pflanzen  transpiration. 

Beih.  Bot.  Cent.  32  :  36. 
Johnston,  E.  S.     1919.     Evaporation  compared  with  vapor-pressure  deficit  and    wind 

velocity.     Mo.  Weather  Rev.  47  :  30. 
Knight,  R.  C.     1915.     A  convenient  modification  of  the  porometer.    New  Phyt.  14  :  212. 

.     1916.     On  the  use  of  the  porometer  in  stomatal  investigation.     Ann.  Bot.  30  :  57. 

.     1917.     "Relative  transpiration"  as  a  measure  of  the  intrinsic  transpiring  power 

of  the  plant.     Ann.  Bot.  31  :  351. 
.     1917  A.     The  interrelations  of  stomatal  aperture,  leaf  water  content,  and  trans- 
piration rate.     Ann.  Bot.  31  :  221. 
Kohl,    F.    G.     1895.     Zum    Mechanik    der    Spaltoflfnungsbewegungen.     Beiblatt    zur 

"Leopoldina." 
Laidlow,  C.  J.  p.,  and  R.  C.  Knight.     1916.    A  description  of  a  recording  porometer 

and  a  note  on  stomatal  behavior  during  wilting.     Ann.  Bot.  30  :  47. 
Leitgkb,   H.     1888.     Beitrage  zur  Physiologic  der  Spaltoffnungsapparate.     Graz   Bot. 
Inst.  Mitt.  1:123. 

103 


BIBLIOGRAPHY.  104 

Livingston,  B.  E.     1906.     Relation  of  desert  plants  to  soil  moisture  and  evaporation. 
Carnegie  Inst.  Wash.  Pub.  No.  50. 
1907.     Relative  transpiration  in  cacti.     Plant  World  10  :  110. 

1909.  Stomata  and  transpiration  in  Tradescantia  zebrina.     Science  29  :  269. 

1910.  Operation  of  porous  cup  vaporimeter.     Plant  World  13  :  111. 

1911.  Light  intensity  and  transpiration.     Bot.  Gaz.  52  :  417. 
1913.     Resistance  offered  by  leaves  to  transpirational  water  loss.     Plant  World 

16  :1. 

.     1915.     Atmometry  and  the  porous  cup.     Plant  World  18  :  21. 

and  W.  H.  Brown.     1912.     Relation  of  the  daily  march  of  transpiration  to  vari-- 

ations  in  the  water  content  of  foliage  leaves.     Bot.  Gaz.  53  :  305. 
and  A.  H.  Estabrook.     1912.     Observations  on  the  degree  of  stomatal  movement 

in  certain  plants.     Bull.  Torr.  Bot.  Club  39:15. 
and  J.  W.  Shive.     1914.     Relation  of  atmospheric  evaporating  power  to  the 

soil-moisture  content  at  permanent  wilting  in  plants.  Plant  World  17  :  81. 
Lloyd,  F.  E.  1908.  The  physiology  of  stomata.  Carnegie  Inst.  Wash.  Pub.  No.  82. 
.     1912.     The  relation  of  transpiration  and  stomatal  movement  to  water  content 

of  leaves  in  Fouquieria  splendens.     Plant  World  15  :  1. 
.     1913.     Leaf  water  and  stomatal  movement  in  Gossypium,  and  a  method  of 

direct  visual  observation  of  stomata  in  situ.  Bull.  Torr.  Bot.  Club  40  : 1. 
Merget,  a.  E.     1873.    Recherches  sur  le  role  des  stomates  dans  les  ph^nomenes  d'echanges 

gazeux  entre  la  plante  et  I'atmosphdre.    Ann.  Bot.  Soc.  Lyon  1:1. 
MoHL,    H.     1856.     Welche   Ursachen   bewirken   die   Erweiterung   und   Verengung   der 

Spaltoffnungen?    Bot.  Zeit.  14:697. 
MoLiscH,  H.     1912.     Das  Offen-  imd  Geschlossensem  der  Spaltoffnungen  veransehaulicht 

durch  eine  neue  Methode.     Zeit.  Bot.  4. 
Mttenscher,  Walter  L.  C.     1915.     Relation  of  transpiration  to  stomata.     Jour.  Bot. 

2  :  487. 
PoRSCH,  — .     1905.     Der  Spaltoffnungsapparat  im  Lichte  Phylogenie. 
Renner,  O.     1910.     Beitrage  zur  Physik  der  Transpiration.     Flora  100  :  451. 

.     1911.     Zur  Physik  der  Transpiration.     Ber.  Deut.  Bot.  Ges.  29  :  451. 

.     1911.     Experimentale    Beitrjige  zur    Kenntniss   der  Wasserbewegung.      Flora 

103  :  171. 
Schwendener,  S.     1881.     Ueber  Bau  und  Mechanik  der  Spaltoffnungen.    Monatsber. 

BerUner  Akad. 
Shreve,  E.  B.  1916.     An  analysis  of  causes  of  variations  of  the  transpiring  power  of 

cacti.     Physiol.  Res.  2  :  73. 
.     1919.     A  thermo-electrical  method  for  the  determination  of  leaf  temperature. 

Plant  World  22:100. 
.     1919a.     The  role  of  temperature  in  the  determination  of  the  transpiring  power 

of  leaves  by  hygrometric  paper.     Plant  World  22:172. 
Stahl,  E.     1894.     Einige  Versuche  iiber  Transpiration   und  Assimilation.     Bot.   Zeit. 

52:117. 
Thomas,    Nesta,   and   Allan   Ferguson.     1917.     On   the   reduction   of   transpiration 

observations.     Ann.  Bot.  122  :  241. 
Trelease,  S.  F.,  and  B.  E.  Livingston.     1916.     The  daily  march  of  transpiring  power 

as   indicated   by   porometer   and  standardized  hygrometric   paper.     Jour. 

Ecol.  4:1. 
Wiggins,  R.  J.     1921.     Variations  in  the  osmotic  concentrations  of  the  guard-cells  during 

opening  and  closing  of  stomata.     Am.  Jour.  Bot.  8  :  30. 


LOFTFIELD 


A.  Wheat  stoma  wedged  open  by  dust,  X  oOO. 

B.  Stoma  of  Rumex  patientia,  X  500. 

C.  Epiderm  of  potato  showing  stomata  in  all  stages  of  development,  X  345. 


LOFTFIELD 


A.  Upper  and  lower  epidcrm  of  alfalfa,  X  345. 

B.  Upper  and  lower  epiderm  of  potato,  X  345. 


LOFTFIELD 


Series  10,  showing  condition  of  upper  (outer)  and  lower  (inner)  stoniata  of  potato,  X  240,  at  each  hour 
of  a  24-hour  day,  together  with  curves  for  sunhght,  temperature,  and  humidity. 


LOFTFIELD 


A.  Upper  and  lower  epiderm  of  sugar-beet. 

B.  Epiderm  of  onion.     C.  Epiderm  of  corn. 


LOFTFIELD 


I 


A.  Cross-section  of  alfalfa  leaf,  showing  stoma  and  chamber,  X  500. 

B.  Cross-section  of  sugar-beet  leaf,  showing  stomata  and  chambers,  X  34o. 


LOFTFIELD 


A.  Cross-section  of  potato  leaf ,  showing  stomata,  chambers,  and  air-passage-^,  X  345. 

B.  Cross-section  of  corn  leaf,  showing  stomata  and  chamljcrs,  X  345. 


LOFTFIELD 


Series  11,  showing  upper  and  lower  stomata  of  sugar-beet,  X  240,  and  factors  during  a  24-iiour  day. 


LOFTFIELD 


PLATE  8 


Series  16,  showino-  stoniat.i  of  oiiion,  yv  24U,  and  factors  during  a  24-hour  dav. 


LOFTFIELD 


Series  16,  showing  stoniata  of  corn,  X  240,  and  factors  during  a  21-liour  dav. 


LOFTFIELD 


LOFTFIELD 


Type  of  jKitoinetor  used  for  stomata  cxpcriniont.' 


LOFTFIELD 


Series  28,  showing  upper  and  lower  stomata  of  Fouqaiera  spleadcns,  X  240,  and  factors  during  a  24-hour  day. 


LOFTFIELD 


PLATE    13 


Series  32,  showing  upper  and  lower  stomata  of  alfalfa,  X  -10,  from  a  heavily  irrigated  plot,  and  factors 

during  a  24-hour  day. 


PLATE  14 


Series  32,  showing  upper  and  lower 


stomata  <:.f  cut  «tems  of  alfalfa,  X  240,  and  factors  during  a  24-hour  day. 


LOFTFIELD 


.•series  33,  j^howiiig  iiijpcr  and  lower  ^^tomata  of  cow-hoct  in  dry  i^ots,  and  factors  during  a  24-hour  day. 


PLATE  16 


Series  33 


,  showing  upper  and  lower  stomata  of  cow-beet  in  moist  soil,  and  factors  during  a  24-hour  day. 


t 


